Therapeutic compounds for blocking dna synthesis of pox viruses

ABSTRACT

This invention provides methods of inhibiting replication of a poxvirus by contacting a poxvirus with a compound having formula I, formula XXI, formula XXXII, or formula XLI which in turn reduce, inhibit, or abrogate poxvirus DNA polymerase activity and/or its interaction with its processivity factor. Formula I, formula XXI, formula XXXII, or formula XLI can be utilized to treat humans and animals suffering from a poxvirus infection. Pharmaceutical compositions for treating poxvirus infected subjects are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to international patent applicationPCT/US08/01553, filed Feb. 6, 2008, which claims priority to U.S.provisional patent applications 60/899,633 and 60/929,673, filed Feb. 6,2007 and Jul. 9, 2007, respectively, all of which are incorporatedherein by reference in their entirety.

GOVERNMENT INTEREST STATEMENT

This invention was made in whole or in part with government supportunder Grant Number U54 AI 57168, awarded by the National Institutes ofHealth. The government may have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods and compositions of preventinginfection by PDX virus using compounds that interfere viral DNAreplication.

BACKGROUND OF THE INVENTION

Poxviruses are the largest known animal viruses. They are DNA virusesthat replicate entirely in the cytoplasm. The 191-kbp genome is adouble-stranded DNA molecule whose ends are covalently connected bysingle-stranded hairpin loops of 101 nucleotides. The sequences thatform the hairpins are AT-rich and lie at the ends of 12-kbp invertedterminal repetition elements that contain short direct repeats andseveral open reading frames.

Poxviruses infect most vertebrates and invertebrates, causing a varietyof diseases of veterinary and medical importance. The one large family(Poxyiridae) has two main subfamilies, the chordopoxyirinae, whichinfect vertebrates, and the entomopoxyirinae, which infect insects.Humans are the sole hosts of two poxviruses, variola virus (smallpoxvirus) and molluscum contagiosum virus, although many members ofOrthopoxvirus, Parapoxvirus, and Yatapoxvirus can infect both animalsand humans. Vaccinia virus is the virus used in the variola virusvaccine, and it is widely used as a model poxvirus.

At least two variants of variola virus are known, and they cause twoforms of smallpox: variola major, with a case fatality rate of 30-40%,and variola minor, with a much reduced fatality rate of about 1%. At thegenome level, the two variants are very similar. Thus, essentially allof the encoded proteins are nearly identical.

Essential viral enzymes have frequently proven to be good targets forantiviral drugs (for example, HIV reverse transcriptase and protease).

The E9 DNA polymerase, required for DNA replication, acts in concertwith accessory proteins to attain efficient processive synthesis.Accessory proteins include the A20 protein, D4R, and others. The viralDNA polymerase is an established drug target, as exemplified byazidothymidine (AZT), which inhibits the HIV reverse transcriptase, andacyclovir, which is efficiently phosphorylated by the herpes simplexvirus viral thymidine kinase, resulting in a triphosphate thatpreferentially inhibits viral DNA polymerase.

Naturally occurring variola virus has been eradicated from the planet.Given the virulence of this virus and its ability to spread in apopulation, the consequences of an intentional release of variola viruscould be devastating. Official stocks of the virus are closely held, butit is not known whether undeclared stocks exist, so it is difficult toassess the current degree of risk. Safer vaccines and therapeutics thatcan mitigate the consequences of infection would together provide astrong deterrent to any intentional release.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus withviridicatumtoxin (formula (XLIV), NSC 55636, NSC 123526, or acombination thereof whereby viridicatumtoxin, NSC 55636, NSC 123526, ora combination thereof reduces, inhibits, or abrogates activity of apoxvirus DNA polymerase.

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus with acompound having the formula I:

A-X—B  I

-   -   wherein A is:        -   Q is NCH₂CH₂R or O;        -   R is OH, N(CH₃)₂ or CH₃;        -   R₁ and R₂ are independently, hydrogen, CH₃, OH or Cl;        -   R₄, R₄′ and R₄″ are independently, hydrogen, COOH, OH, CF₃,            Cl, Br, COOMe, OMe, N(CH₃)₂ or NO₂;        -   W₁ is alky, alkyl-isothiourea or substituted alkyl;        -   B is 1 of the following structures:

-   -   -   R₃ and R₃′ are hydrogen, COOH, OH, COOMe, Cl, CF₃, CH₃,            OCH₃, N(CH₃)₂ or CN;        -   W₂ is alkyl, alky-isothiourea or substituted alkyl, —SO2Et,            H or isopropyl;        -   W₃ is 2, 4 dimethoxy phenyl;        -   W₄ is CH₃ or NH—W₃; and        -   P is hydrogen, Fmoc, or Boc;

    -   X is nothing, SO₂, —TeCH₂CH₂NHCH₂CH₂Te—, NH, S,

—CO—, —CH₂S—, —N═CH— —COO—, —OCO—,

-   -   or A-X—B are fused rings, wherein X is a 5-membered substituted        or non-substituted heterocyclic or carbocyclic, optionally        aromatic ring represented by 1 of the following structures:

-   -   A is

and

-   -   B is

-   -   whereby the compound reduces, inhibits, or abrogates activity of        a poxvirus DNA polymerase.

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus with acompound having the formula XXI, whereby the compound reduces, inhibits,or abrogates activity of a poxvirus DNA polymerase.

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus with acompound having the formula XXXII, whereby the compound reduces,inhibits, or abrogates activity of a poxvirus DNA polymerase.

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus with acompound having the formula XLI, whereby the compound reduces, inhibits,or abrogates activity of a poxvirus DNA polymerase.

In one embodiment, this invention provides a method of inhibitingreplication of a poxvirus, comprising contacting a poxvirus with amixture comprising compounds having the formula XXI, XXXII, XLI, wherebythe compound reduces, inhibits, or abrogates activity of a poxvirus DNApolymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingadministering to a subject viridicatumtoxin, NSC 55636, NSC 123526, or acombination thereof whereby viridicatumtoxin, NSC 55636, NSC 123526, ora combination thereof reduces, inhibits, or abrogates activity of apoxvirus DNA polymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingadministering to the subject a compound having the formula I:

A-X—B  I

-   -   wherein A is 1 of the following structures:

-   -   Q is NCH₂CH₂R or O;    -   R is OH, N(CH₃)₂ or CH₃;    -   R₁ and R₂ are independently, hydrogen, CH₃, OH, Cl;    -   R₄, R₄′ and R₄″ are independently, hydrogen, COOH, OH, CF₃, Cl,        Br, COOMe, OMe, N(CH₃)₂ or NO₂;        W1 is alkyl, alky-isothiourea or substituted alkyl    -   B is

-   -   R₃ and R₃′ are hydrogen, COOH, OH, COOMe, Cl, CF₃, CH₃, OCH₃,        N(CH₃)₂ or CN;    -   W₂ is alkyl, alky-isothiourea or substituted alkyl, —SO2Et, H or        isopropyl;    -   W₃ is 2, 4 dimethoxy phenyl;    -   W₄ is CH₃ or NH—W₃; and    -   P is hydrogen, Fmoc, or Boc;        -   X is nothing, NH, S,

—CO—, —CH2S—, —N═CH— —COO—, —OCO—,

or A-X—B are fused rings, wherein X is a 5-membered substituted or notsubstituted heterocyclic or carbocyclic, optionally aromatic ringrepresented by the following structures:

-   -   A is

and

-   -   B is

whereby the compound reduces, inhibits, or abrogates activity of apoxvirus DNA polymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingcontacting a poxvirus with a compound having the formula XXI, wherebythe compound reduces, inhibits, or abrogates activity of a poxvirus DNApolymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingcontacting a poxvirus with a compound having the formula XXXII, wherebythe compound reduces, inhibits, or abrogates activity of a poxvirus DNApolymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingcontacting a poxvirus with a compound having the formula XLI, wherebythe compound reduces, inhibits, or abrogates activity of a poxvirus DNApolymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingcontacting a poxvirus with a mixture comprising compounds having theformula XXI, XXXII, XLI, whereby the compound reduces, inhibits, orabrogates activity of a poxvirus DNA polymerase.

In another embodiment, this invention provides a method of inhibiting,treating, or abrogating a poxvirus infection in a subject, comprisingcontacting a poxvirus with a compound having the formula XLIV, wherebythe compound reduces, inhibits, or abrogates activity of a poxvirus DNApolymerase.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of formula XLIV.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from a reading of the followingdetailed description taken in conjunction with the drawings in whichlike reference designators are used to designate like elements, and inwhich:

FIG. 1. Flow-chart of stepwise screening protocol. In Step I, the NCIDiversity and Training Sets were screened using a lysate fromvaccinia-infected cells. In Step II, the primary hits were screened toeliminate general and irrelevant inhibitors using the KSHV Pol8/PF8 DNAsynthesis plate assay. In Step III, compounds were then tested on twodistinct plate assays to differentiate polymerase from processiveinhibitors. DNA synthesis inhibition by the hits was confirmed using anM13 assay (Step IV). The final four hits were tested for viral plaquereduction (Step V) and cellular cytotoxicity (Step VI). The screenyielded a polymerase and a processivity inhibitor of vaccinia virus DNAsynthesis.

FIG. 2. A typical example of primary rapid plate assay result. Compoundsthat inhibited DNA synthesis by more than 50% are indicated by solidbars, and those that inhibit DNA synthesis by less than 50% areindicated by shaded bars. The (+) symbols indicate positive controlinhibitor EDTA and the (−) symbol indicate negative control inhibitorsunphosphorylated AZT, unphosphorylated acyclovir, DMSO alone and nocompound added.

FIG. 3. Assay to eliminate general and irrelevant inhibitors. Hitcompounds from the primary screen using vaccinia lysates were furthertested in a KSHV rapid plate assay for their abilities to inhibit DNAsynthesis directed by Pol8/PF8. Compounds that inhibited Pol8/PF8 DNAsynthesis (solid bars) were categorized as general or irrelevantinhibitors. Compounds that failed to inhibit Pol8/PF8 DNA synthesis(shaded bars) were considered to be bonafide inhibitors of vaccinia DNAsynthesis. The (+) symbol indicates positive control inhibitor EDTA andthe (−) symbol indicates DMSO alone and no compound added.

FIG. 4. Assay to distinguish polymerase and processive inhibitors ofvaccinia DNA synthesis. (A) The model depicts the uniform incorporationof the DIG-dUTP on a template by E9 polymerase under low saltconditions. Under low salt conditions, E9 incorporates dNTPs onto theDNA template to completion. (B) NCI hit compounds were analyzed on theuniform template in the presence of E9 alone under low salt conditionsto identify polymerase inhibitors. Compounds 69343 and 55636 blockedpolymerase activity whereas 123526 and 124808 had minimal effects. (C)The model depicts incorporation of the label DIG-dUTP on the distal endof the template by the triad (A20, D4, E9) under high salt conditions.Under high salt conditions, E9 requires A20 and D4 to accomplishprocessive DNA synthesis. (D) NCI hit compounds were analyzed on thedistal tempate in the presence of the triad under high salt conditionsto identify processivity inhibitors. Compounds 124808 and 123526 failedto block dNTP incorporation on the uniform template but blockedincorporation on the distal template. Compounds that block E9 polymeraseactivity also blocked processivity. For both A and C, the template 5′end is biotinylated for attachment to the streptavidin-coated plates.

FIG. 5. Confirmation of inhibitors using the M13 DNA synthesis assay.Full-length ds DNA products labeled with [α-³²P]dCTP were synthesizedfrom the M13 DNA primer/template in the presence of the vaccinia triad(A20, D4, E9) and examined on a non-denaturing gel. The ability of eachpolymerase and processivity inhibitor to block the M13 DNA synthesis wasexamined by adding increasing concentrations (100 nM, 1 μM, 10 μM, 100μM and 1 mM) of each compound: NSC 69343, lanes 3-7; NSC 55636, lanes8-12; NSC 123526, lanes 13-17; NSC 124808, lanes 18-22. Lane 1 shows theposition of the full-length ds M13 DNA marker (arrow) as detected bycybergold. Lane 2 is the control reaction containing DMSO, the solventfor each of the compounds. The position of greater than unit lengthproducts are indicated by brackets.

FIG. 6. Analysis of vaccinia DNA synthesis inhibitors by plaquereduction and cell cytotoxicity assays. The solid bars represent thereduction of plaques in the presence of inhibitors (100 μM) relative tono inhibitor. The shaded bars represent the percent cell cytotoxicity inthe presence of inhibitors (100 μM) relative to no inhibitor. A minimumof six repetitions were performed for the cytotoxicity and the plaquereduction assays.

FIG. 7. Purity of the cytoplasmic extract of vaccinia virus-infectedcells. (A) Western blot. Cytoplasmic and nuclear extracts of BSC-1 cellsinfected with vaccinia virus were analyzed by Western blot withmonoclonal antibodies against the cytoplasmic marker β-Actin and thenuclear marker Rb-1. (B) DNA synthesis activities of cytoplasmicextracts in rapid plate assay. Lane 1: Reaction catalyzed by uninfectedcytoplasmic extract. Lane 2: Reaction catalyzed by vaccinia-infectedcytoplasmic extract. Lane 3: Reaction catalyzed by vaccinia-infectedcytoplasmic extract that was completely inhibited with EDTA.

FIG. 8. Dose-dependent inhibition of vaccinia virus DNA synthesis byselected compounds. (IC50's in legend next to compound name.)

FIG. 9. Representative Class I and Class I tetracycline molecules.

FIG. 10. Inhibition of vaccinia E9 polymerase-directed DNA synthesis byviridicatumtoxin and tetracycline. DNA synthesis was conducted in therapid plate assay using low-salt conditions that favor extended strandsynthesis of vaccinia E9 polymerase produced by in vitro translation.DNA synthesis activity was measured by the incorporation of DIG-dUTPinto the newly synthesized strand. Increasing amounts ofviridicatumtoxin () and tetracycline (▴) were added to the reactions,from which the (IC50) of each compound was determined.

FIG. 11. Reduction of vaccinia virus plaques by viridicatumtoxin andtetracycline. Vaccinia virus infected BSC-1 cells were treated withincreasing concentrations of viridicatumtoxin () and tetracycline (▴)at one hour post infection. The cells were fixed, stained and the numberof plaques were determined. The concentration of each compound requiredto reduce plaques by 50% (IC₅₀) was determined.

FIG. 12. Effect of viridicatumtoxin and tetracycline on vaccinia earlyand late viral mRNA expression. Total RNA was isolated from BSC-1 cellsthat were infected for 8 hr with vaccinia virus in the presence of 20 μMviridicatumtoxin or tetracycline. Vaccinia early gene E3 (A) and lategene F9 (B) were quantitated by RT-PCR and normalized to the levels ofcellular graph. The data represents the average of duplicateexperiments.

FIG. 13. Effect of viridicatumtoxin and tetracycline on vaccinia earlyand late viral protein expression. Lysates were prepared at differenttime points from BSC-1 cells that were infected with vaccinia virus inthe presence of 20 μM viridicatumtoxin or tetracycline. The lysates wereanalyzed by western blot using vaccinia early E3 or late L1 proteinantibodies as probes. The signal obtained with the GAPDH antibody servedas an internal standard. Cells infected with DMSO alone served as anegative control.

FIG. 14. Inhibition of vaccinia virus DNA synthesis and antiviralactivity of 3 typical hit compounds. (A) Inhibition of vaccinia virusDNA synthesis by each compound was tested in triplicate over a range ofconcentrations in the DNA synthesis rapid plate assay. Percentinhibition was calculated relative to the uninhibited negative control(DMSO) and the completely inhibited positive control (EDTA). The Prismsoftware was used to plot the inhibitory dose-response and to calculatethe IC50 values. (B) Antiviral activity measured in the cell protectionassay. The ability of compounds to block vaccinia virus infection inBS-C-1 cells was measured over a wide range of concentrations. Twentyhours after infection, cells were fixed with formaldehyde, stained withcrystal violet, and the extent of antiviral protection was quantified bymeasuring absorbance at 570 nm. Every compound was tested in triplicateand the percent protection was calculated relative to the DMSO anduninfected controls. The Prism software was used to plot the protectiondose—response and to calculate the EC50 values.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of inhibiting, treating, orabrogating a poxvirus infection in a subject; inhibiting replication ofa poxvirus; inhibiting activity of a poxvirus DNA polymerase; anddecreasing processivity of a poxvirus DNA polymerase, comprisingcontacting a poxvirus with a compound of the present invention.

In another embodiment, the compound described herein isviridicatumtoxin. In another embodiment, viridicatumtoxin of the presentinvention has the following formula:

In another embodiment, viridicatumtoxin reduces, inhibits, or abrogatesactivity of a poxvirus DNA polymerase. In

In another embodiment, viridicatumtoxin reduces, inhibits, or abrogatesinteraction of a poxvirus DNA polymerase with its processivity factor.

In another embodiment, the compound as described herein is NSC 55636. Inanother embodiment, the compound NSC 55636 has the following formula:

In another embodiment, NSC 55636 reduces, inhibits, or abrogatesinteraction of a poxvirus DNA polymerase with its processivity factor.

In another embodiment, the compound as described herein is NSC 123526.In another embodiment, the compound NSC 123526 has the followingformula:

In another embodiment, NSC 123526 reduces, inhibits, or abrogatesinteraction of a poxvirus DNA polymerase with its processivity factor.

In certain embodiments, the compound as described herein has thestructure of formula I:

A-X—B  I

wherein A is:

or nothing

Q is NCH₂CH₂R or O; R is OH, N(CH₃)₂ or CH₃;

R1 and R2 are independently, hydrogen, CH₃, OH, Cl;R4, R4′ and R4″ are independently hydrogen, COOH, OH, CF₃, Cl, Br,COOMe, OMe, N(CH₃)₂ or NO₂;W1 is alkyl, alky-isothiourea or substituted alkyl;

B is

R₃ and R₃′ are hydrogen, COOH, OH, COOMe, Cl, CF₃, CH₃, OCH₃, N(CH₃)₂ orCN;W2 is alkyl, alky-isothiourea or substituted alkyl, —SO2Et, H orisopropyl;W3 is 2, 4 dimethoxy phenyl;

W4 is CH3 or NH—W3; and

P is hydrogen, Fmoc, or Boc;X is nothing, SO₂, —TeCH₂CH₂NHCH₂CH₂Te—, NH, S,

—CO—, —CH2S—, —N═CH—,

—COO—, —OCO—,

or A-X—B are fused rings, wherein X is a 5-membered substituted or notsubstituted heterocyclic or carbocyclic, optionally aromatic ringrepresented by one of the following structures:

A is

and

B is

whereby the compound reduces, inhibits, or abrogates activity of apoxvirus DNA polymerase. In another embodiment, the compound reduces,inhibits, or abrogates interaction of a poxvirus DNA polymerase with itsprocessivity factor.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

wherein R1 is phenyl substituted or non substituted by OH orCH=(heterocyclic 5-10 membered ring);R₂ is O, NH, NR, wherein R is CH2CH2X;

X is OH, CH₃, N(CH₃)₂; and

R₃ and R₄ are independently H, Cl, CH₃ or OH.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith viridicatumtoxin of the present invention.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith NSC 55636 of the present invention.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith NSC 123526 of the present invention.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the heterocyclic 5-10 membered ring of formula IIis:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

wherein A and B are independently hydrogen or they form a bond;

X is NH, Net, C═NOH, CR₃R₄;

R1′ and R1 are independently CF₃, COOH, COOCH₃, CH₃, Cl, OCH₃, OH orCH═N-triazole;R2′ and R₂ are independently CF₃, COOH, COOCH₃, CH₃, Cl, OCH₃, OH orCH═N-triazole; andR3 and R4 are independently chlorobenzene or SCH₂C(COOH)NH₂.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxyir uswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of one of the following formulas:

or a mixture thereof.

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, the present invention provides a method ofinhibiting replication of a poxvirus, comprising contacting a poxviruswith a compound of the formula:

In another embodiment, viridicatumtoxin as described herein issynthesized by P. viridicatum. In another embodiment, viridicatumtoxinas described herein is synthesized by methods known to one skilled inthe art.

In another embodiment, viridicatumtoxin as described herein has apoxvirus plaque IC50 of 400-1000 nM. In another embodiment,viridicatumtoxin as described herein has a poxvirus plaque IC50 of400-500 nM. In another embodiment, viridicatumtoxin as described hereinhas a poxvirus plaque IC50 of 450-600 nM. In another embodiment,viridicatumtoxin as described herein has a poxvirus plaque IC50 of500-750 nM. In another embodiment, viridicatumtoxin as described hereinhas a poxvirus plaque IC50 of 680-780 nM. In another embodiment,viridicatumtoxin as described herein has a poxvirus plaque IC50 of780-1000 nM. In another embodiment, viridicatumtoxin as described hereinhas a poxvirus plaque IC50 of 750 nM. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, NSC 55636 as described herein has a poxvirusplaque IC50 of 400-1000 nM. In another embodiment, NSC 55636 asdescribed herein has a poxvirus plaque IC50 of 400-500 nM. In anotherembodiment, NSC 55636 as described herein has a poxvirus plaque IC50 of450-600 nM. In another embodiment, NSC 55636 as described herein has apoxvirus plaque IC50 of 500-750 nM. In another embodiment, NSC 55636 asdescribed herein has a poxvirus plaque IC50 of 680-780 nM. In anotherembodiment, NSC 55636 as described herein has a poxvirus plaque IC50 of780-1000 nM.

In another embodiment, NSC 123526 as described herein has a poxvirusplaque IC50 of 400-1000 nM. In another embodiment, NSC 123526 asdescribed herein has a poxvirus plaque IC50 of 400-500 nM. In anotherembodiment, NSC 123526 as described herein has a poxvirus plaque IC50 of450-600 nM. In another embodiment, NSC 123526 as described herein has apoxvirus plaque IC50 of 500-750 nM. In another embodiment, NSC 123526 asdescribed herein has a poxvirus plaque IC50 of 680-780 nM. In anotherembodiment, NSC 55636 as described herein has a poxvirus plaque IC50 of780-1000 nM.

In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 130 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 200 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 150 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 170 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 190 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 210 μM.In another embodiment, viridicatumtoxin as described herein has apoxvirus cell cytotoxicity-50 value (CC50) that is greater than 230 μM.

In one embodiment, viridicatumtoxin, is an effective inhibitor ofvaccinia virus. In another embodiment, viridicatumtoxin interferes withvaccinia E9 DNA polymerase activity. In yet another embodiment,viridicatumtoxin reduces plaques at near nanomolar concentrations (IC₅₀of 1.6 μM).

In one Viridicatumtoxin is not cytotoxic even when cells are exposed to200 μM. In another embodiment, as described hereinbelow, the therapeuticindex of viridicatumtoxin is greater than 125. In another embodiment,the therapeutic index of viridicatumtoxin is significantly higher thanthe therapeutic index of the DNA synthesis inhibitor cidofovir.

In one embodiment viridicatumtoxin prevents viral gene expressionwithout affecting early viral genes. In another embodiment,viridicatumtoxin is nonteratogenic when orally administered to pregnantmice during mid-gestation.

In one embodiment, viridicatumtoxin, but not tetracycline, contains aspirocyclohexene moiety that imparts specificity through its bulk andstereochemistry. In another embodiment, viridicatumtoxin inhibitsnucleotide incorporation in vivo

In one embodiment, viridicatumtoxin is a novel non-nucleoside inhibitorof vaccinia virus DNA synthesis that is capable of blocking infection atconcentrations that are not toxic to cells. In another embodiment,viridicatumtoxin is a used as a substrate for generating new and morepotent poxvirus inhibitors.

In another embodiment, NSC 123526 as described herein has a poxviruscell cytotoxicity-50 value (CC₅₀) that is greater than 130 μM. Inanother embodiment, NSC 123526 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 150 μM. In anotherembodiment, NSC 123526 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 170 μM. In anotherembodiment, NSC 123526 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 190 μM. In anotherembodiment, NSC 123526 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 210 μM. In anotherembodiment, NSC 123526 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 230 μM.

In another embodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 130 μM. In anotherembodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 150 μM. In anotherembodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 170 μM. In anotherembodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 190 μM. In anotherembodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 210 μM. In anotherembodiment, NSC 55636 as described herein has a poxvirus cellcytotoxicity-50 value (CC₅₀) that is greater than 230 μM.

In another embodiment, viridicatumtoxin as described herein has apoxvirus therapeutic index greater than 180. In another embodiment,viridicatumtoxin as described herein has a poxvirus therapeutic indexgreater than 200. In another embodiment, viridicatumtoxin as describedherein has a poxvirus therapeutic index greater than 215. In anotherembodiment, viridicatumtoxin as described herein has a poxvirustherapeutic index greater than 240. In another embodiment,viridicatumtoxin as described herein has a poxvirus therapeutic indexgreater than 250. In another embodiment, viridicatumtoxin as describedherein has a poxvirus therapeutic index greater than 270. In anotherembodiment, viridicatumtoxin as described herein has a poxvirustherapeutic index greater than 280.

In another embodiment, NSC 55636 as described herein has a poxvirustherapeutic index greater than 180. In another embodiment, NSC 55636 asdescribed herein has a poxvirus therapeutic index greater than 200. Inanother embodiment, NSC 55636 as described herein has a poxvirustherapeutic index greater than 215. In another embodiment, NSC 55636 asdescribed herein has a poxvirus therapeutic index greater than 240. Inanother embodiment, NSC 55636 as described herein has a poxvirustherapeutic index greater than 250. In another embodiment, NSC 55636 asdescribed herein has a poxvirus therapeutic index greater than 270. Inanother embodiment, NSC 55636 as described herein has a poxvirustherapeutic index greater than 280.

In another embodiment, NSC 123526 as described herein has a poxvirustherapeutic index greater than 180. In another embodiment, NSC 123526 asdescribed herein has a poxvirus therapeutic index greater than 200. Inanother embodiment, NSC 123526 as described herein has a poxvirustherapeutic index greater than 215. In another embodiment, NSC 123526 asdescribed herein has a poxvirus therapeutic index greater than 240. Inanother embodiment, NSC 123526 as described herein has a poxvirustherapeutic index greater than 250. In another embodiment, NSC 123526 asdescribed herein has a poxvirus therapeutic index greater than 270. Inanother embodiment, NSC 123526 as described herein has a poxvirustherapeutic index greater than 280.

In another embodiment, viridicatumtoxin is administered orally. Inanother embodiment, viridicatumtoxin is administered intraperitoneally(i.p.). In another embodiment, viridicatumtoxin is administeredsubcutaneously (s.c.) routes.

In another embodiment, NSC 123526 is administered orally. In anotherembodiment, NSC 123526 is administered intraperitoneally (i.p.). Inanother embodiment, NSC 123526 is administered subcutaneously (s.c.).

In another embodiment, NSC 55636 is administered orally. In anotherembodiment, NSC 55636 is administered intraperitoneally (i.p.). Inanother embodiment, NSC 55636 is administered subcutaneously (s.c.).

In another embodiment, the mice s.c. LD₅₀ of a compound as describedherein are at least 100 mg/kg body weight. In another embodiment, themice s.c. LD₅₀ of a compound as described herein are at least 150 mg/kgbody weight. In another embodiment, the mice s.c. LD₅₀ of a compound asdescribed herein are at least 200 mg/kg body weight. In anotherembodiment, the mice s.c. LD₅₀ of a compound as described herein are atleast 250 mg/kg body weight. In another embodiment, the mice s.c. LD₅₀of a compound as described herein are at least 300 mg/kg body weight. Inanother embodiment, the mice s.c. LD₅₀ of a compound as described hereinare at least 350 mg/kg body weight. In another embodiment, the mice s.c.LD₅₀ of a compound as described herein are at least 380 mg/kg bodyweight. In another embodiment, the mice s.c. LD₅₀ of a compound asdescribed herein are at least 400 mg/kg body weight. In anotherembodiment, the mice s.c. LD₅₀ of a compound as described herein are atleast 450 mg/kg body weight.

In certain embodiments, the poxvirus as described herein infectsvertebrates. In certain embodiments, the poxvirus as described hereininfects invertebrates. In certain embodiments, the poxvirus of thepresent causes a variety of diseases of veterinary and medicalimportance. In certain embodiments, the poxvirus as described hereinbelongs to the chordopoxyirinae subfamily. In another embodiment, thepoxvirus as described herein is variola virus (smallpox virus). Inanother embodiment, the poxvirus is vaccinia virus. In anotherembodiment, the poxvirus is molluscum contagiosum virus. In otherembodiments, the poxvirus is any known orthopoxvirus, parapoxvirus, oryatapoxvirus.

In another embodiment, the poxvirus is a cowpox virus. In anotherembodiment, the poxvirus is a monkeypox virus. In another embodiment,the poxvirus is a raccoonpox virus. In another embodiment, the poxvirusis a camelpox virus. In another embodiment, the poxvirus is a skunkpoxvirus. In another embodiment, the poxvirus is a volepox virus. Inanother embodiment, the poxvirus is an ectromelia virus. In anotherembodiment, the poxvirus is a taterapox virus.

In another embodiment, the poxvirus is a parapoxvirus. In anotherembodiment, the poxvirus is an orf virus. In another embodiment, thepoxvirus is a pseudocowpox virus. In another embodiment, the poxvirus isany other type of parapoxvirus known in the art.

In another embodiment, the poxvirus is an avipoxvirus. In anotherembodiment, the poxvirus is a canarypox virus. In another embodiment,the poxvirus is a fowlpox virus. In another embodiment, the poxvirus isany other type of avipoxvirus known in the art.

In another embodiment, the poxvirus is a capripoxvirus. In anotherembodiment, the poxvirus is a goatpox virus. In another embodiment, thepoxvirus is a lumpy skin disease virus. In another embodiment, thepoxvirus is any other type of capripoxvirus known in the art.

In another embodiment, the poxvirus is a leporipoxvirus. In anotherembodiment, the poxvirus is a myxoma virus. In another embodiment, thepoxvirus is a fibroma virus. In another embodiment, the poxvirus is anyother type of leporipoxvirus known in the art.

In another embodiment, the poxvirus is a molluscipoxvirus. In anotherembodiment, the poxvirus is a molluscum contagiosum virus. In anotherembodiment, the poxvirus is any other type of molluscipoxvirus known inthe art.

In another embodiment, the poxvirus is a yatapoxvirus. In anotherembodiment, the poxvirus is a tanapox virus. In another embodiment, thepoxvirus is a Yaba monkey tumor virus. In another embodiment, thepoxvirus is any other type of yatapoxvirus known in the art.

In another embodiment, the poxvirus is any other type of poxvirus knownin the art. In another embodiment, each of the above poxviruses andtypes of poxviruses represents a separate embodiment of the presentinvention.

In certain embodiments, methods of inhibiting replication of a poxviruscomprise methods of inhibiting the DNA thereof. In certain embodiments,inhibiting the DNA replication is achieved by inhibiting activity of aDNA polymerase protein. In certain embodiments, inhibiting a DNApolymerase protein activity comprises reducing the processivity of a DNApolymerase. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the DNA polymerase that is inhibited is an E9protein. In another embodiment, the DNA polymerase is a variola DNApolymerase. In another embodiment, the DNA polymerase has a sequence setforth in 1 of the following GenBank Accession Numbers: DQ437580;DQ437581; DQ437582; DQ437583-92, inclusive; DQ441416-48, inclusive.

In certain embodiments, DNA polymerase protein processive activity isinhibited in the presence of an accessory protein. In anotherembodiment, interaction of a DNA polymerase with an accessory protein isinhibited or reduced. In another embodiment, interaction of a DNApolymerase with a processivity factor is inhibited or reduced. Inanother embodiment, an E9 DNA polymerase processivity accessory proteinor processivity factor is a stoichiometric component of the processiveform of poxvirus DNA polymerase. In another embodiment, the accessoryprotein is an A20 protein.

In another embodiment, the accessory protein is a D4R (D4; UDG). Inanother embodiment, the accessory protein is a D5 gene product. Inanother embodiment, the accessory protein is an H5 gene product. Inanother embodiment, the accessory protein is a homologue of A20 fromanother species. In another embodiment, the accessory protein is ahomologue of D4 from another species. In another embodiment, theaccessory protein is a homologue of D5 from another species. In anotherembodiment, the accessory protein is a homologue of H5 from anotherspecies. In another embodiment, the accessory protein is any otherpoxvirus DNA polymerase accessory protein known in the art. Eachpossibility represents a separate embodiment of the present invention.

In certain embodiments, the poxvirus E9 DNA polymerase protein is atleast 70% homologous to a vaccinia virus E9 DNA polymerase proteinsequence. In another embodiment, the homology is at least 75%. Inanother embodiment, the homology is at least 80%. In another embodiment,the homology is at least 85%. In another embodiment, the homology is atleast 88%. In another embodiment, the homology is at least 90%. Inanother embodiment, the homology is at least 92%. In another embodiment,the homology is at least 95%. In another embodiment, the homology is atleast 97%. In another embodiment, the homology is at least 98%.

In another embodiment, the E9 DNA polymerase protein is a variola virusE9 DNA polymerase protein. In another embodiment, the E9 DNA polymeraseprotein is at least 80% homologous to variola virus E9 DNA polymeraseprotein. In another embodiment, the homology is at least 85%. In anotherembodiment, the homology is at least 88%. In another embodiment, thehomology is at least 90%. In another embodiment, the homology is atleast 92%. In another embodiment, the homology is at least 95%. Inanother embodiment, the homology is at least 97%. In another embodiment,the homology is at least 98%.

In certain embodiments, the poxvirus E9 DNA polymerase processivityaccessory protein is at least 70% homologous to vaccinia virus A20protein sequence. In another embodiment, the homology is at least 75%.In another embodiment, the homology is at least 80%. In anotherembodiment, the homology is at least 85%. In another embodiment, thehomology is at least 88%. In another embodiment, the homology is atleast 90%. In another embodiment, the homology is at least 92%. Inanother embodiment, the homology is at least 95%. In another embodiment,the homology is at least 97%. In another embodiment, the homology is atleast 98%.

In another embodiment, the poxvirus E9 DNA polymerase processivityaccessory protein is an A20 variola virus processivity accessoryprotein. In another embodiment, the poxvirus E9 DNA polymeraseprocessivity accessory protein is at least 80% homologous to variolavirus A20 protein sequence. In another embodiment, the homology is atleast 85%. In another embodiment, the homology is at least 88%. Inanother embodiment, the homology is at least 90%. In another embodiment,the homology is at least 92%. In another embodiment, the homology is atleast 95%. In another embodiment, the homology is at least 97%. Inanother embodiment, the homology is at least 98%.

In certain embodiments, the poxvirus E9 DNA polymerase processivityaccessory protein is at least 70% homologous to vaccinia virus D4Rprotein sequence. In another embodiment, the homology is at least 75%.In another embodiment, the homology is at least 80%. In anotherembodiment, the homology is at least 85%. In another embodiment, thehomology is at least 88%. In another embodiment, the homology is atleast 90%. In another embodiment, the homology is at least 92%. Inanother embodiment, the homology is at least 95%. In another embodiment,the homology is at least 97%. In another embodiment, the homology is atleast 98%.

In another embodiment, the poxvirus E9 DNA polymerase processivityaccessory protein is a D4R variola virus processivity accessory protein.In another embodiment, the poxvirus E9 DNA polymerase processivityaccessory protein is at least 80% homologous to a variola virus D4Rprotein sequence. In another embodiment, the homology is at least 85%.In another embodiment, the homology is at least 88%. In anotherembodiment, the homology is at least 90%. In another embodiment, thehomology is at least 92%. In another embodiment, the homology is atleast 95%. In another embodiment, the homology is at least 97%. Inanother embodiment, the homology is at least 98%.

In certain embodiments, contacting a poxvirus with a compound asdescribed herein comprises the step of adding the compound to a petridish comprising cells infected with a poxvirus. In certain embodiments,contacting a poxvirus with a compound as described herein comprisesadding the compound to a petri dish comprising an organ culture infectedwith a poxvirus. In certain embodiments, contacting a poxvirus with acompound as described herein comprises administering the compound to ananimal and/or subject infected with a poxvirus.

In certain embodiments, a compound as described herein is solubilized ina buffer compatible with the media comprising cells or a tissue culture.In another embodiment, a compound as described herein is solubilized inthe media comprising cells or a tissue culture. In certain embodiments,a compound as described herein is suspended or otherwise emulsified bymethods known to one skilled in the art.

In certain embodiments, the present invention provides methods ofinhibiting, a poxvirus infection in an animal and/or subject comprisingadministering to an animal and/or subject a compound of the presentinvention. In certain embodiments, the term inhibiting comprisesrestraining, holding back, repressing, or preventing.

In another embodiment, a compound utilized in methods as describedherein has an EC₅₀ for a poxvirus of 4.8 μM. In another embodiment, theEC₅₀ is 0.05 μM. In another embodiment, the EC₅₀ is 0.1 μM. In anotherembodiment, the EC₅₀ is 0.15 μM. In another embodiment, the EC₅₀ is 0.2μM. In another embodiment, the EC₅₀ is 0.3 μM. In another embodiment,the EC50 is 0.4 μM. In another embodiment, the EC₅₀ is 0.5 μM. Inanother embodiment, the EC₅₀ is 0.7 μM. In another embodiment, the EC50is 1 μM. In another embodiment, the EC₅₀ is 1.5 μM. In anotherembodiment, the EC₅₀ is 2 μM. In another embodiment, the EC₅₀ is 3 μM.In another embodiment, the EC₅₀ is 5 μM. In another embodiment, the EC₅₀is 7 μM. In another embodiment, the EC₅₀ is 10 μM. In anotherembodiment, the EC₅₀ is 15 μM. In another embodiment, the EC₅₀ is 16.1μM. In another embodiment, the EC₅₀ is 20 μM. In another embodiment, theEC₅₀ is 30 μM. In another embodiment, the EC₅₀ is 50 μM. In anotherembodiment, the EC₅₀ is 70 μM.

In another embodiment, the EC₅₀ is 0.1-30 μM. In another embodiment, theEC₅₀ is 0.1-1 μM. In another embodiment, the EC₅₀ is 0.1-2 μM. Inanother embodiment, the EC₅₀ is 0.1-3 μM. In another embodiment, theEC₅₀ is 0.1-5 μM. In another embodiment, the EC₅₀ is 0.1-7 μM. Inanother embodiment, the EC₅₀ is 0.1-10 μM. In another embodiment, theEC₅₀ is 0.1-15 μM. In another embodiment, the EC₅₀ is 0.1-20 μM. Inanother embodiment, the EC₅₀ is 0.2-1 μM. In another embodiment, theEC₅₀ is 0.2-2 μM. In another embodiment, the EC₅₀ is 0.2-3 μM. Inanother embodiment, the EC₅₀ is 0.2-5 μM. In another embodiment, theEC₅₀ is 0.2-7 μM. In another embodiment, the EC₅₀ is 0.2-10 μM. Inanother embodiment, the EC₅₀ is 0.2-15 μM. In another embodiment, theEC₅₀ is 0.2-20 μM. In another embodiment, the EC₅₀ is 0.3-1 μM. Inanother embodiment, the EC₅₀ is 0.3-2 μM. In another embodiment, theEC₅₀ is 0.3-3 μM. In another embodiment, the EC₅₀ is 0.3-5 μM. Inanother embodiment, the EC₅₀ is 0.3-7 μM. In another embodiment, theEC₅₀ is 0.3-10 μM. In another embodiment, the EC₅₀ is 0.3-15 μM. Inanother embodiment, the EC₅₀ is 0.3-20 μM. In another embodiment, theEC₅₀ is 0.5-1 μM. In another embodiment, the EC₅₀ is 0.5-2 μM. Inanother embodiment, the EC₅₀ is 0.5-3 μM. In another embodiment, theEC₅₀ is 0.5-5 μM. In another embodiment, the EC₅₀ is 0.5-7 μM. Inanother embodiment, the EC₅₀ is 0.5-10 μM. In another embodiment, theEC₅₀ is 0.5-15 μM. In another embodiment, the EC₅₀ is 0.5-20 μM. Inanother embodiment, the EC₅₀ is 1-2 μM. In another embodiment, the EC₅₀is 1-3 μM. In another embodiment, the EC₅₀ is 1-5 μM. In anotherembodiment, the EC₅₀ is 1-7 μM. In another embodiment, the EC₅₀ is 1-10μM. In another embodiment, the EC₅₀ is 1-15 μM. In another embodiment,the EC₅₀ is 1-20 μM. In another embodiment, the EC₅₀ is 2-3 μM. Inanother embodiment, the EC₅₀ is 2-5 μM. In another embodiment, EC₅₀ is2-7 μM. In another embodiment, the EC₅₀ is 2-10 μM. In anotherembodiment, the EC₅₀ is 2-15 μM. In another embodiment, the EC₅₀ is 2-20μM. In another embodiment, the EC₅₀ is 2-30 μM. In another embodiment,the EC50 is 2-50 μM. In another embodiment, the EC₅₀ is 3-5 μM. Inanother embodiment, the EC₅₀ is 3-7 μM. In another embodiment, the EC₅₀is 3-10 μM. In another embodiment, the EC₅₀ is 3-15 μM. In anotherembodiment, the EC₅₀ is 3-20 μM. In another embodiment, the EC₅₀ is 3-30μM. In another embodiment, the EC₅₀ is 3-50 μM. In another embodiment,the EC₅₀ is 5-7 μM. In another embodiment, the EC₅₀ is 5-10 μM. Inanother embodiment, the EC₅₀ is 5-15 μM. In another embodiment, the EC₅₀is 5-20 μM. In another embodiment, the EC₅₀ is 5-30 μM. In anotherembodiment, the EC₅₀ is 5-50 μM. In another embodiment, the EC₅₀ is 7-10μM. In another embodiment, the EC₅₀ is 7-15 μM. In another embodiment,the EC₅₀ is 7-20 μM. In another embodiment, the EC₅₀ is 7-30 μM. Inanother embodiment, the EC₅₀ is 7-50 μM. In another embodiment, the EC₅₀is 10-12 μM. In another embodiment, the EC₅₀ is 10-15 μM. In anotherembodiment, the EC₅₀ is 10-20 μM. In another embodiment, the EC₅₀ is10-30 μM. In another embodiment, the EC₅₀ is 10-50 μM. In anotherembodiment, the EC₅₀ is 15-20 μM. In another embodiment, the EC₅₀ is15-30 μM. In another embodiment, the EC₅₀ is 15-40 μM. In anotherembodiment, the EC₅₀ is 15-50 μM.

Each of the above values for EC₅₀ represents a separate embodiment ofthe present invention.

In certain embodiments, the present invention provides methods oftreating a poxvirus infection in an animal and/or subject comprisingadministering to an animal and/or subject a compound of the presentinvention.

In certain embodiments, the present invention provides methods ofabrogating a poxvirus infection in an animal and/or subject comprisingadministering to an animal and/or subject a compound of the presentinvention. In certain embodiments, the term abrogating comprisesabolishing or terminating.

In certain embodiments, the compounds of this invention are formulatedinto a pharmaceutical dosage form. In certain embodiments, thepharmaceutical dosage form further comprises pharmaceutically acceptablecarriers, excipients, emollients, stabilizers, etc., as are known in thepharmaceutical industry. In some embodiments the pharmaceutical dosagewill include other active agents such immune system modifiers. Inanother embodiment, other compounds for stabilizing, preserving, theformulation and the like, but are not involved directly in thetherapeutic effect of the indicated active ingredient, are included.

In certain embodiments, the pharmaceutical compositions containing thecompounds as described herein are administered to a subject by anymethod known to a person skilled in the art, such as parenterally,paracancerally, transmucosally, transdermally, intramuscularly,intravenously, intradermally, subcutaneously, intraperitonealy,intraventricularly, intracranially, intravaginally or intratumorally.

Various embodiments of dosage ranges are contemplated by this invention.In one embodiment, the dosage of the compounds as described herein is inthe range of 0.1-100 mg/day. In another embodiment, the dosage is in therange of 0.1-50 mg/day. In another embodiment, the dosage is in therange of 0.1-20 mg/day. In another embodiment, the dosage is in therange of 0.1-10 mg/day. In another embodiment, the dosage is in therange of 0.1-5 mg/day. In another embodiment, the dosage is in the rangeof 0.5-5 mg/day.

If the preferred mode is administered orally, in another embodiment, aunit dosage form comprises tablets, capsules, lozenges, chewabletablets, suspensions, emulsions and the like. In certain embodiments,such unit dosage forms comprise a safe and effective amount of thedesired compound, or compounds, each of which is in another embodiment,from about 0.5 or 10 mg to about 300 mg/70 kg, or in another embodiment,about 0.5 or 10 mg to about 210 mg/70 kg. In certain embodiments, thepharmaceutically-acceptable carrier suitable for the preparation of unitdosage forms for peroral administration is well-known in the art. Incertain embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmelose; lubricants such as magnesium stearate,stearic acid and talc. In certain embodiments, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In certain embodiments, coloring agents, such as theFD&C dyes, can be added for appearance. In certain embodiments,sweeteners and flavoring agents, such as aspartame, saccharin, menthol,peppermint, and fruit flavors, are useful adjuvants for chewabletablets. In certain embodiments, capsules typically comprise one or moresolid diluents disclosed above. In certain embodiments, the selection ofcarrier components depends on secondary considerations like taste, cost,and shelf stability, which are not critical for the purposes of thisinvention, and can be readily made by a person skilled in the art.

In certain embodiments, peroral compositions comprise liquid solutions,emulsions, suspensions, and the like. In certain embodiments, thepharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. In certain embodiments, liquidoral compositions comprise, in certain embodiments, from about 0.012% toabout 0.933% of the desired compound or compounds, or in anotherembodiment, from about 0.033% to about 0.7%.

In another embodiment, the dosage is 10-20 μg/tablet. In anotherembodiment, the dosage is 20-30 μg/tablet. In another embodiment, thedosage is 20-40 μg/tablet. In another embodiment, the dosage is 30-60μg/tablet. In another embodiment, the dosage is 40-80 μg/tablet. Inanother embodiment, the dosage is 50-100 μg/tablet. In anotherembodiment, the dosage is 50-150 μg/tablet. In another embodiment, thedosage is 100-200 μg/tablet. In another embodiment, the dosage is200-300 μg/tablet. In another embodiment, the dosage is 300-400μg/tablet. In another embodiment, the dosage is 400-600 μg/tablet. Inanother embodiment, the dosage is 500-800 μg/tablet. In anotherembodiment, the dosage is 800-1000 μg/tablet. In another embodiment, thedosage is 1000-1500 μg/tablet. In another embodiment, the dosage is1500-2000 μg/tablet. In another embodiment, the dosage is 2-3 mg/tablet.In another embodiment, the dosage is 2-5 mg/tablet. In anotherembodiment, the dosage is 2-10 mg/tablet. In another embodiment, thedosage is 2-20 mg/tablet. In another embodiment, the dosage is 2-30mg/tablet. In another embodiment, the dosage is 2-50 mg/tablet. Inanother embodiment, the dosage is 2-80 mg/tablet. In another embodiment,the dosage is 2-100 mg/tablet. In another embodiment, the dosage is 3-10mg/tablet. In another embodiment, the dosage is 3-20 mg/tablet. Inanother embodiment, the dosage is 3-30 mg/tablet. In another embodiment,the dosage is 3-50 mg/tablet. In another embodiment, the dosage is 3-80mg/tablet. In another embodiment, the dosage is 3-100 mg/tablet. Inanother embodiment, the dosage is 5-10 mg/tablet. In another embodiment,the dosage is 5-20 mg/tablet. In another embodiment, the dosage is 5-30mg/tablet. In another embodiment, the dosage is 5-50 mg/tablet. Inanother embodiment, the dosage is 5-80 mg/tablet. In another embodiment,the dosage is 5-100 mg/tablet. In another embodiment, the dosage is10-20 mg/tablet. In another embodiment, the dosage is 10-30 mg/tablet.In another embodiment, the dosage is 10-50 mg/tablet. In anotherembodiment, the dosage is 10-80 mg/tablet. In another embodiment, thedosage is 10-100 mg/tablet.

In one embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in another embodimentare aqueous solutions or emulsions comprising a safe and effectiveamount of a compound as described herein and in yet another embodiment,other compounds. In one embodiment, such compositions comprise fromabout 0.01% to about 10.0% w/v of a subject compound, more preferablyfrom about 0.1% to about 5.0, which in another embodiment, is used forthe systemic delivery of compounds by a route known to one skilled inthe art.

In certain embodiments, the compositions comprise dry powders. Incertain embodiments, compositions are formulated for atomization and/orinhalation administration. In certain embodiments, such compositions arecontained in a container with attached atomizing means.

Further, in another embodiment, the pharmaceutical compositions areadministered by intravenous, intra-arterial, or intramuscular injectionof a liquid preparation. In certain embodiments, suitable liquidformulations include solutions, suspensions, dispersions, emulsions,oils and the like. In another embodiment, the pharmaceuticalcompositions are administered intravenously, and are thus formulated ina form suitable for intravenous administration. In another embodiment,the pharmaceutical compositions are administered intra-arterially, andare thus formulated in a form suitable for intra-arterialadministration. In another embodiment, the pharmaceutical compositionsare administered intramuscularly, and are thus formulated in a formsuitable for intramuscular administration.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In another embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989). In another embodiment, polymeric materials can be used.In yet one embodiment, a controlled release system can be placed inproximity to the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990).

In certain embodiments, the preparation of pharmaceutical compositionswhich contain active components is well understood in the art, forexample by mixing, granulating, or tablet-forming processes. In certainembodiments, the active therapeutic ingredients are mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. In certain embodiments, for oral administration, thecompounds as described herein or their physiologically toleratedderivatives such as salts, esters, N-oxides, and the like and additionaltherapeutic agent or agents are mixed with additives customary for thispurpose, such as vehicles, stabilizers, or inert diluents, and convertedby customary methods into suitable forms for administration, such astablets, coated tablets, hard or soft gelatin capsules, aqueous,alcoholic or oily solutions.

In certain embodiments, an active component as described herein isformulated into the composition as neutralized pharmaceuticallyacceptable salt forms. In certain embodiments, pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule), which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. In certain embodiments, salts formed from the free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium, or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

In certain embodiments, for use in medicine, the salts of the compoundsas described herein will be pharmaceutically acceptable salts. Incertain embodiments, other salts may, however, be useful in thepreparation of the compounds used in the methods described herein, or oftheir pharmaceutically acceptable salts. In certain embodiments,suitable pharmaceutically acceptable salts of the compounds of thisinvention include acid addition salts which may, for example, be formedby mixing a solution of the compound according to the invention with asolution of a pharmaceutically acceptable acid such as hydrochloricacid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid,succinic acid, acetic acid, benzoic: acid, oxalic acid, citric acid,tartaric acid, carbonic acid or phosphoric acid.

In certain embodiments, the compositions also comprise preservatives,such as benzalkonium chloride and thimerosal and the like; chelatingagents, such as edetate sodium and others; buffers such as phosphate,citrate and acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acids and bases to adjustthe pH of these aqueous compositions as needed. In certain embodiments,the compositions may also comprise local anesthetics or other actives.In certain embodiments, the compositions can be used as sprays, mists,drops, and the like.

In certain embodiments, substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. In certain embodiments, thechoice of a pharmaceutically-acceptable carrier to be used inconjunction with the compound is basically determined by the way thecompound is to be administered. In certain embodiments, wherein thesubject compound is to be injected, the preferredpharmaceutically-acceptable carrier is sterile, physiological saline,with a blood-compatible suspending agent, the pH of which has beenadjusted to about 7.4.

In certain embodiments, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants.

In certain embodiments, typical components of carriers for syrups,elixirs, emulsions and suspensions include ethanol, glycerol, propyleneglycol, polyethylene glycol, liquid sucrose, sorbitol and water. For asuspension, typical suspending agents include methyl cellulose, sodiumcarboxymethyl cellulose, cellulose (e.g. AVICEL™, RC-591), tragacanthand sodium alginate; typical wetting agents include lecithin andpolyethylene oxide sorbitan (e.g. polysorbate 80). In certainembodiments, typical preservatives include methyl paraben and sodiumbenzoate. In certain embodiments, peroral liquid compositions alsocontain one or more components such as sweeteners, flavoring agents andcolorants disclosed above.

In certain embodiments, dry powder compositions may comprise propellantssuch as chlorofluorocarbons 12/11 and 12/114, or, in another embodiment,other fluorocarbons, nontoxic volatiles; solvents such as water,glycerol and ethanol, these include co-solvents as needed to solvate orsuspend the active; stabilizers such as ascorbic acid, sodiummetabisulfite; preservatives such as cetylpyridinium chloride andbenzalkonium chloride; tonicity adjustors such as sodium chloride;buffers; and flavoring agents such as sodium saccharin.

In certain embodiments, the compositions also include incorporation ofthe active material into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.) Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance.

In certain embodiments, also comprehended by the invention areparticulate compositions coated with polymers (e.g. poloxamers orpoloxamines) and the compound coupled to antibodies directed againsttissue-specific receptors, ligands or antigens or coupled to ligands oftissue-specific receptors.

In certain embodiments, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Themodified compounds are known to exhibit substantially longer half-livesin blood following intravenous injection than do the correspondingunmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; andKatre et al., 1987). In certain embodiments, such modifications may alsoincrease the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In certain embodiments, the desired in vivo biologicalactivity may be achieved by the administration of such polymer-compoundabducts less frequently or in lower doses than with the unmodifiedcompound.

In certain embodiments, the compounds of the invention can beadministered as the sole active pharmaceutical agent, they can also beused in combination with one or more other compound as described herein,and/or in combination with other agents used in the treatment and/orprevention of diseases, disorders and/or conditions, associated with apoxvirus infection, as will be understood by one skilled in the art. Inanother embodiment, the compounds as described herein can beadministered sequentially with one or more such agents to providesustained therapeutic and prophylactic effects. In another embodiment,the compounds may be administered via different routes, at differenttimes, or a combination thereof. It is to be understood that any meansof administering combined therapies which include the compounds of thisinvention are to be considered as part of this invention.

In another embodiment, the additional active agents are generallyemployed in therapeutic amounts as indicated in the PHYSICIANS' DESKREFERENCE (PDR) 53rd Edition (1999), which is incorporated herein byreference, or such therapeutically useful amounts as would be known toone of ordinary skill in the art. In another embodiment, the compoundsof the invention and the other therapeutically active agents areadministered at the recommended maximum clinical dosage or at lowerdoses. In certain embodiments, dosage levels of the active compounds inthe compositions of the invention may be varied to obtain a desiredtherapeutic response depending on the route of administration, severityof the disease and the response of the patient. In another embodiment,the combination is administered as separate compositions or in otherembodiments as a single dosage form containing both agents. In certainembodiments, when administered as a combination, the therapeutic agentsis formulated, in another embodiment, as separate compositions that aregiven at the same time or different times, or in other embodiments thetherapeutic agents can be given as a single composition.

In certain embodiments, the compositions and methods described hereinare employed in the treatment of domesticated mammals which aremaintained as human companions (e.g., dogs, cats, horses), which havesignificant commercial value (e.g., dairy cows, beef cattle, sportinganimals), which have significant scientific value (e.g., captive or freespecimens of endangered species), or which otherwise have value.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Materials and Methods Rapid Plate Assay

The rapid mechanistic plate assay, a high-throughput screening of achemical library of chemical compounds, was used to identify inhibitorsof processive DNA synthesis. The vaccinia virus (VV) E9 DNA polymeraseprotein and the processive VV proteins A20 and, D4R were used as targetsfor the inhibitors screened.

The following materials were used:

-   1. Cytoplasmic extract of vaccinia infected BS-C-1 cells.-   2. 20-mer Oligonucleotide primer 5′-GCCAATGAATGACCGCTGAC-3′ (SEQ ID    No. 1).-   3. 5′ Biotinylated 100-mer oligonucleotide template: 5′    biotin-GCACTTATTGCATTCGCTAGTCCACCTTGGATCTCAGGCTATTCGTAGCGACCTA    CGCGTACGTTAGCTTCGGTCATCCCGTCAGCGGTCATTCATTGGC-3′ (SEQ ID No. 2).-   4. Streptavidin-coated, transparent, nuclease-free plates (Roche).-   5. DIG-dUTP (digoxigenin-11-2′-deoxy-uridine-5′-triphosphate),    alkali-stable (Roche).-   6. DIG Detection ELISA (ABTS, Roche) that contains    antidigoxigenin/peroxidase, Fab fragments conjugated with peroxidase    (POD). POD reacts with the substrate ABTS    (2,2′-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid])-   7. Phosphate-buffered saline (PBS), 10× stock solution. Per liter:    80 g NaCl, 2 g KCl, 11.5 g Na2HPO4.7H2O, and 2 g KH2PO4.-   8. PBS working solution, pH 7.3: 137 mM NaCl, 2.7 mM KCl, 4.3 mM    Na2HPO4, and 1.4 mM KH2PO4.-   9. Wash buffer: PBS and 0.1% Tween-20.-   10. Blocking stock solution (10×): Dissolve blocking reagent (Roche)    in maleic acid buffer by constant stifling on a heating block (65°    C.) or heat in a microwave, autoclave, and store at 4° C. The    solution remains opaque.-   11. PBS/1% blocking solution: Dilute the blocking stock solution    (10×) 1:10 in PBS to 1%, and store at 4° C.-   12. Maleic acid buffer: dissolve 10.6 g maleic acid (0.1 M) and 8.76    g NaCl (0.15 M) in 900 mL dH2O, and adjust pH to 7.5 with 10 N NaOH.    Add ddH2O to 1 L.-   13. Preparation of premix solution for DNA synthesis according to    Table 1.-   14. Addition of 1 μL each of in vitro—synthesized E9, A20, and D4.

TABLE 1 Preparation of Premix Solution for DNA Synthesis^(a) Amount ofstock Final Stock solution (μL) Component conc. solution per well(NH₄)₂SO₄ 100 mM 1M 5.0 Tris-HCl, pH 7.5 20 mM 200 mM 5.0 MgCl₂ 3 mM 100mM 1.5 EDTA 0.1 mM 5 mM 1.0 DTT 0.5 mM 10 mM 2.5 Glycerol 4% 50% 4.0 BSA40 μg/mL 10 mg/mL 0.2 dATP 50 μM 5 mM 0.5 dGTP 50 μM 5 mM 0.5 dCTP 50 μM5 mM 0.5 DIG-dUTP 10 μM 1 mM 0.5 BSA = bovine serum albumin; DIG =digoxigenin; DTT = dithiothreitol.

The Rapid Plate Assay Overview: A template with biotin attached to its5′-end and a primer annealed to its 3′-end was bound to thestreptavidin-coated wells of 96-well plates. The premix solution,containing vaccinia cytoplasmic extract and dNTPs with DIG-dUTP(substituted for dTTP), was then added to the wells of the plates. Thiswas followed by the addition of the chemical test compounds. The plateswere incubated to enable the DNA synthesis reaction to proceed. Then thereaction was stopped, and the incorporation of dNTPs into synthesizedDNA was detected by an ELISA reaction that employs anti-DIG antibodyconjugated to peroxidase.

Annealing Primer to Template:

250 μmol (1.525 μg) of primer and 250 μmol (8.25 μg) of biotinylatedtemplate were mixed in 0.25 mL PBS (pH 7.3), and annealed by heating to90° C. for 5 minutes, followed by cooling to room temperature. Then theannealed primer/template (P/T) was diluted to 0.1 μM with cold PBS, andstored at −20° C.

Binding Primed Template to 96-Well Plates:

0.2 μmol of P/T (1.22 ng of primer and 6.6 ng of template) in 100 μLwere added to each well of the streptavidin-coated plates, followed byincubation of the foil-covered wells for 90 min at 37° C. or overnightat 4° C.

DNA Synthesis Reaction:

The P/T binding solution was removed from the 96-well plates and 25.2 μLof the premix solution was added to each well, followed by the additionof the chemical test compound or H₂O (24.8 μL) to a final reactionvolume of 50 μL 2. 3. Then the reaction mixture was incubated at 37° C.for 90 min. For a negative control, a separate premix solution was usedin which E9, A20, and D4R were absent. The reaction was stopped byadding 1 μL of 0.5 M EDTA.

First Plate Wash:

The reaction mixture was removed, and wells were washed six times with200, 225, 250, 275, 300, and 325 μL of wash buffer.

Binding of Anti-DIG-POD:

100 μL anti-DIG-POD working solution (final concentration of 200 mU/mLin PBS/1% blocking reagent) was added followed by incubation at 37° C.for 1 h.

Second Plate Wash:

The anti-DIG-POD working solution was removed followed by washing sixtimes with 200, 225, 250, 275, 300, and 325 μL of wash buffer.

POD Color Detection as a Measure of DNA Synthesis:

The ABTS substrate for POD was added to the wells. Color developmentgenerally occurred within 5-30 min to produce an OD 405 nm of 0.4-1.0.

Quantification of DNA Synthesis by ELISA:

ELISA reader at 405 nm was used to quantify absorbance proportional tothe incorporated DIG-dUTP.

Compound Library

The NCI Training and Diversity Set libraries were obtained from the DrugSynthesis and Chemistry Branch, Developmental Therapeutics Program,National Cancer Institute. The Training Set contains 230 anti-cancercompounds that are frequently used to confirm the reproducibility ofhigh throughput screening assays. The Diversity Set comprises 1,992compounds selected from approximately 140,000 compounds using the Chem-Xprogram (Accelrys, San Diego, Calif., USA).

These compounds represent diversity in terms of three-dimensionalpharmacophores.

In Vitro Translated Proteins and Vaccinia Virus-Infected Cell Lysate

Vaccinia virus polymerase (E9), putative processivity factor (A20) andUDG (D4) proteins were expressed from pcDNA3.2/v5 (Invitrogen) in vitrousing Promega TNT coupled transcription/translation system. KSHV DNApolymerase-8 (Pol8) and processivity factor-8 (PF8) were translated invitro from pTM1-Pol8 and pTM1-PF8 respectively. To confirm expression,an aliquot of the translation reactions were labeled with[³⁵S]-methionine, fractionated on an SDS-10% polyacrylamide gel, andvisualized by autoradiography. The vaccinia virus-infected cell lysatewas prepared according to previously described methods. The WR strain ofvaccinia virus, a thymidine kinase deficient mutant, was used to infectmonolayers of BSC-1 cells at a multiplicity of infection of 15. Thevaccinia-infected cells were incubated at 37° C. for 6 h in the presenceof hydroxyurea. The cells were harvested by scraping then pelleted at500 rpm. The pellet was washed with phosphate-buffered saline (PBS)followed by hypotonic buffer (10 mM Hepes, 1.5 nM MgCl₂, 10 mM KCl).After resuspension in hypotonic buffer, the cells were Douncehomogenized and centrifuged at 15,000 rpm for 30 min. The suspension waspassed through a 2 micron filter to remove the viral cores and nuclearparticles. The vaccinia-infected lysate was stored in −80° C. in thepresence of 20% glycerol.

High-Throughput Screening for Inhibitors of DNA Synthesis Using theRapid Plate Assay

A rapid plate DNA synthesis assay was performed using vaccinia-infectedcell lysate. A 1.2:1 ratio of a 20-mer oligonucleotide primer(5′-GCGAATGAATGACCGCTGAC-3′, SEQ ID No. 1) and a 5′-end biotinylated100-mer oligonucleotide template (5′-Biotin-GCACTTATTGCATTCGCTAGTCCACCTTGGATCTCAGGCTATTCGTAGCGAGCTACGCGTACGTTAGCTTCGGTCATCCCGTCAGCGGTCATTCATTGGC-3′, SEQ ID No. 2) were heated at 90° C. for 5 min andannealed by gradual cooling to room temperature. The annealedprimer-template (P/T) was diluted with PBS to a concentration of 10μmol/μL. The 96-well microtiter streptavidin-coated plates (Streptawellplates, Roche Applied Science, Indianapolis, Ind., USA) were coated with5 μmol/well of the P/T solution and incubated at 37° C. for 90 min. Thewells were washed with 100 μL PBS. Control (DMSO, acyclovir prodrug,azidothymidine (AZT) prodrug and ethylenediamine tetracetic acid (EDTA))and test compounds were individually added to the wells to a finalconcentration of 167 μM. The vaccinia virus used in this primary screenis a thymidine kinase deficient mutant, and therefore resistant to AZTand acyclovir, prodrugs which require phosphorylation. The 60 μL DNAsynthesis reaction mixture contained 100 mM (NH₄)₂SO₄, 20 mM Tris-HCl pH7.4, 3 mM MgCl₂, 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP,Roche Applied Science) and 1 μL vaccinia lysate. The plates wereincubated at 37° C. for 30 min. Total DNA synthesis activities weredetermined by measuring incorporation of DIG-dUTP. The activity wasquantified by a DIG detection ELISA kit (Roche Applied Science) usinganti-digoxigenin-peroxidase (anti-DIG-POD) and its substrate2,2′-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS), and by measuringthe absorbance at 405 nm on a microplate reader (Tecan Genius Pro, TecanUS).

Selectivity Screen

The selectivity screen was used to eliminate general and irrelevantinhibitors of vaccinia DNA synthesis. The microplate assay was optimizedfor KSHV Pol8/PF8 DNA synthesis. P/T solution of 0.2 pmols was added toeach well. Each reaction well received control or test compounds and 2μL each of in vitro translated Pol8 and PF8. The reaction time wasextended to 1 h at 37° C. The buffer, BSA and dNTP conditions used werethe same as described for vaccinia, vide supra.

Polymerase vs. Processivity Inhibition

Two separate plate assays were used to distinguish polymerase versusprocessivity inhibitors. For the polymerase inhibition assay, abiotinylated primer/template (5′-GCGAATGAATGACCGCTGAC-3′, SEQ ID No.1)/(5′-Biotin-GCACTTATTGCATTCGCTAGTCCACCTTGGATCTCAGGCTATTCGTAGCGAGCTACGCGTACGTTAGCTTCGGTCATCCCGTCAGCGGTCATTCATTGGC-3′ SEQ ID No. 2)was designed so that the DIG epitope would be uniformly incorporatedthroughout the template. The DNA synthesis reaction was performed usinga low salt buffer (20 mM Tris-HCl pH 7.4, 3 mM MgCl₂, 0.1 mM EDTA, 0.5mM DTT, and 2% glycerol) and 1 μL polymerase enzyme E9. For theprocessivity inhibition assay, a second biotinylated primer/template(5′-GCCAATGAATGACCGCTGAC-3′)/(5′-Biotin-AGCACTATTGACATTACAGAGTCGCCTTGGCTCTCTGGCTGTTCGTTGCGGGCTCCGCGTGCGTTGGCTTCGGTCGTCCCGTCAGCGGTCATTCATTGGC-3′) was designed so that theDIG epitope would be incorporated at the distal end of the template. Theplate assay was conducted using a high salt buffer (100 mM (NH₄)₂SO₄, 20mM Tris-HCl pH 7.4, 3 mM MgCl₂, 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol).One microliter each of the in vitro translated proteins A20, D4, and E9were added to each well.

M13 DNA Synthesis

In vitro DNA synthesis using an M13-primed template was performed.Briefly, the DNA synthesis reactions were performed in 25 μL volumescontaining 10 mM Tris-HCl (pH 7.5), 40 mg/mL of bovine serum albumin, 4%glycerol, 0.1 mM EDTA, 5 mM dithiothreitol (DTT), 8 mM MgCl₂, 20 fmol ofsingly primed M13 mp 18 single-stranded (ss) DNA, 750 ng of E. coli SSB,60 μM (each) dGTP, dTTP and dATP, and 20 μM [α-³²P]dCTP. The mixtureswere pre-incubated with the proteins and dATP, dGTP and dTTP at 30° C.for 3 min. The reactions were initiated by the addition of radiolabeleddCTP, incubated for another 30 min and stopped by the addition of anequi-volume of 1% SDS-40 mM EDTA. The reaction products werefractionated at 60V on a 0.8% denaturing agarose gel and analyzed by aPhosphorImager (Amersham Biosciences).

Plaque Reduction Assay

The cell lines used were African green monkey kidney BSC-1 cells grownin Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetalbovine serum (FBS) (Gibco BRL Life Technologies, Gaithersburg, Md.) and0.1% gentamicin antibiotic. The cell cultures were maintained at 37° C.in a humidified 5% CO₂ environment. Confluent BSC-1 cells were infectedwith 100 μL/well of 100-150 pfu of vaccinia virus in a 48-well plate.After 1 h incubation, 400 μL/well of DNA synthesis inhibitor (100 μM) orcontrol solutions were added and incubated at 37° C. overnight. Allinhibitors and controls were dissolved in DMSO and diluted with themedium. A 5% solution of formaldehyde in PBS was used to fix the cells.After washing twice with PBS, the plate was stained with 0.2% crystalviolet in 50% ethanol.

Cytotoxicity Assay

BSC-1 cells were grown to confluency in white 96-well cell cultureplates at 37° C. in DMEM containing 10% FBS and 0.1% gentamicin in thepresence or absence of inhibitor. Cytotoxicity was assayed using the aCella-Tox bioluminescence cytotoxicity kit (Cell Technology Inc.,Mountainview, Calif.) according to manufacturer's protocol. Allinhibitors and controls were assayed at a final concentration of 100 μM.A lysing agent was used to calculate the maximum release ofglyceraldehyde-3-phosphate dehydrogenase as a positive control.

Compound Confirmation

The molecular weight of the hit compounds were confirmed using lowresolution liquid chromatography/mass spectrometry, electrosprayionization mode (ES+, Micromass LC, Opus software system, Department ofChemistry, University of Pennsylvania).

Primary High-Throughput Screen

The primary high-throughput screen was performed in 384-well platescoated with streptavidin SigmaScreen plates, Sigma-Aldrich cat #S8686).DNA synthesis reactions were run with 0.5 μL vaccinia virus extract in20 mM Tris-Cl pH7.5, 100 mM ammonium sulfate, 5 mM MgCl2, 0.1 mM EDTA,0.5 mM DTT, 4% glycerol, 40 μg/mL BSA, 5 μM of each dATP, dCTP, anddGTP, 4 μM dTTP, 0.5 μM DIG-1′-dUTP, in a total volume of 30 μL. Twopmoles of biotinylated primer/template dissolved in 30 μL PBS wereimmobilized on streptavidin coated wells. Unbound primer/template wasremoved, wells were washed twice with 50 μL PBS and loaded with 20 μLreaction buffer (20 mM Tris-Cl pH7.5). One hundred nL of compound ornatural extract dissolved in DMSO were transferred from the libraryplates with 384-pin arrays. Reactions were started by loading 10 μL of a3× reaction mixture. After quick centrifugation, plates were incubatedat 37 C for 30 minutes. The DNA synthesis reactions were stopped with 30μL of a solution containing 50 mM EDTA and 2% SDS in 10 mM Tris pH8.Incorporation of DIG-11-dUTP in the newly synthesized DNA strand wasmeasured with peroxidase-conjugated anti-digoxigenin antibody(anti-DIGPOD). Wells were flow-washed with 400 μL PBS, 0.1% Tween-20 atthe lowest dispensing speed and loaded with 4.5 mU of anti-DIG-POD in 30μL PBS/blocking solution. After gently rocking at room temperature for 1h, the antibody solution was removed and wells were flow-washed asabove. Thirty μL of 2,2′-azino-bis(3-ethylbenzthiazoline)-sulfonate(ABTS) peroxidase substrate dissolved in citrate buffer was added andplates were gently rocked at room temperature for 1 h. Color developmentwas stopped with 10 uL of 4% SDS and absorbance at 405 nm was measured.Readings from each well were divided by the plate median and percentinhibition was determined relative to the values of uninhibited reactioncontrol.

Screened Libraries

The optimized assay was first trained and confirmed by screening 1,520known bioactive chemical compounds compiled in two collections: BIOMOLICCB Known Bioactives 1 (480 compounds) and NINDS Custom Collection(1,040 compounds). A total of 45,832 chemical compounds from thefollowing libraries: ChemDiv 3 (16,544 compounds), MixCommercial 5 (268compounds), Maybridge 4 (4,576 compounds), ActiMol TimTec 1 (8,518compounds), Bionet 2 (1,700 compounds), Enamine 1 (6,004 compounds),I.F. Lab 1 (6,543 compounds), I.F. Lab 2 (292 compounds), Maybridge 2(704 compounds), MixCommercial 4 (331 compounds), and Peakdale 2 (352compounds) were tested in duplicate, at a single concentration of 16.7μg/mL, which is equivalent to a molar concentration of 33 μM or higher.Natural extracts with inhibitory activity were also identified byscreening the Stan Foundation Extracts 2 library (1,000 extracts fromplants used in traditional Chinese medicine), and two collections ofpartially purified extracts from endophytic fungi: ICBG Fungal Extracts1 (851 extracts) and ICBG Fungal Extracts 2 (460 extracts).

DNA Synthesis Assay

Selected compounds were purchased and suspended in DMSO at a finalconcentration of 20 mM. The compounds were tested over a range ofconcentrations for their ability to inhibit vaccinia virus-catalyzed DNAsynthesis. Assays were run in 96-well plates in conditions similar tothose used in the high-throughput screen. Each compound was tested intriplicate in two-fold serial increments and IC50 values were calculatedusing the Prism software for linear regression.

Antiviral Activity Assays in Cultured Cells (Plaque Reduction Assay andCell Protection Assay)

For the plaque reduction assay, BSC-1 cells were seeded at 6×104cells/well in 48-well plates. Next day, cells were infected with 50plaque forming units (pfu)/well of vaccinia virus WR in DMEM with 10%FBS and 50 mg/L gentamicin sulfate. Tested compounds were diluted ingrowth medium and added over the virus/cell cultures. Plates wereincubated overnight at 37° C. with 5% CO2. Cells were fixed with asolution of 5% formaldehyde in PBS and stained with 0.2% crystal violetdissolved in 50% ethanol. Wells were washed, let dry and plaques werecounted under a microscope. The final concentration of compound-derivedDMSO was 1% (v/v) in all cellbased assays. For the control samples thatcontained no compound, an appropriate volume of DMSO was added at afinal concentration of 1%.

Cytotoxicity Assay Reagents and Materials Proteins

Processive DNA synthesis was catalyzed by early-expressed vacciniaproteins from cytoplasmic extracts of BS-C-1 cells infected with thevaccinia virus strain WR. harvested 6 hours after infection. Thecytoplasmic extracts were filtered twice through 0.2 mm and contained noinfectious particle as shown by plaque assay.

Annealed Primer/Biotinylated Template: synthesized by Integrated DNATechnologies Primer 20 nucleotides ((5′-GCGAATGAATGACCGCTGAC-3′, SEQ IDNo. 1) Template 100 nucleotides 5′ end biotinylated (5′Biotin-GCACTTATTGCATTCGCTAG TCCACCTTGG ATCTCAGGCT ATTCGTAGCG AGCTACGCGTACGTTAGCTT CGGTCATCCC GTCAGCGGTC ATTCATTGGC-3′).

Annealing: 15 nmoles (92 mg) of primer and 15 nmoles (470 mg) oftemplate are mixed in 1.5 mL PBS (pH 7.3), and annealed by heating to 90C for 5 minutes, then cooled to room temperature. The final P/Tconcentration is 10 mM or 10 pmole/mL.

SigmaScreen Streptavidin coated plates; 384-well, clear (Sigmacat#58686-100EA); Digoxigenin-11-2′-deoxy-uridine-5′-triphosphate(DIG-11-dUTP), alkali-stable, 1 mM (Roche cat#11 570 013 910);Deoxynucleotide Triphosphate Set, PCR Grade, Na-Salt, 100 mM (Rochecat#11969064001); Anti-Digoxigenin-POD, Fab fragments from sheep (Rochecat#11 207 733 910); 2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulfonicacid) diammonium salt (ABTS), (Roche cat#10 102 946 001) dissolved inABTS buffer (Roche cat#11 112 597 001) at 1 mg/mL; Blocking reagent(Roche, Cat #11 096 176001) 10% (w/v) in maleic acid buffer (0.1 Mmaleic acid, 0.15 M NaCl, pH to 7.5) diluted 1:10 in PBS to 1% (w/v)final. Stop: 2% SDS, 50 mM EDTA, 10 mM Tris pH8 Wash: PBS+0.1% Tween-20

Analog Compounds

Analogs of tetracycline were obtained through compound mining byperforming structural similarity searches using the Bit VectorStructural Map online http://spheroid.ncifcrf.gov/spheroid/ developed bythe Developmental Therapeutic Program of the National Cancer Instituteto aid in drug discovery. The 52 tetracycline structurally relatedcompounds were obtained from the Drug Synthesis and Chemistry Branch,Developmental Therapeutics Program, National Cancer Institute(http://dtp.nci.nih.gov/).

Vaccinia Virus-Infected Cytoplasmic Lysate and In Vitro TranslatedProteins

Throughout the study, thymidine kinase (TK) deficient strain of vacciniavirus, provided by Drs. G. Cohen and R. Eisenberg, was used to infectBSC-1 cells (34). Vaccinia virus-infected cell lysate was prepared aspreviously described (16). Briefly, the cells were infected at amultiplicity of infection of 15. The vaccinia-infected cells wereincubated at 37° C. for 6 h in the presence of hydroxyurea, thenharvested by scraping and pelleted at 500 rpm. The pellet was washedwith phosphate-buffered saline (PBS) followed by hypotonic buffer (10 mMHepes, 1.5 mM MgCl₂, 10 mM KCl). The cells were then Dounce homogenizedand centrifuged at 15,000 rpm for 30 min. The cell suspension was passedthrough a 2 micron filter to remove the viral cores and nuclearparticles. At this point, the vaccinia-infected cytoplasmic lysate wasstored in −80° C. in the presence of 20% glycerol. Vaccinia E9, A20 andD4 proteins were translated in vitro as previously described (34). Theproteins were expressed from pcDNA3.2/v5 (Invitrogen) in vitro usingPromega TNT coupled transcription/translation system. The translationreactions were labeled with [₃₅S]-methionine, fractionated on 10%SDS-PAGE, and visualized by autoradiography.

DNA Synthesis Inhibition Assay

A rapid plate DNA synthesis assay (19) was performed using optimizedconditions. Briefly, a 1.2:1 ratio of a 20-mer oligonucleotide primer(5′-GCGAATGAATGACCGCTGAC-3′, SEQ ID No. 1) and a 5′-end biotinylated100-mer oligonucleotide template(5′-Biotin-AGCACTATTGACATTACAGAGTCGCCTTGGCTCTCTGGCTGTTCGTTGCGGGCTCCGCGTGCGTTGGCTTCGGTCGTCCCGTCAGCGGTCATTCATTGGC-3′) were annealed and loadedinto a 96-well microtiter streptavidin-coated plate (Streptawell plates,Roche Applied Science, Indianapolis, Ind., USA) at 5 μmol/well. Thewells were incubated at 37 C for 90 min, and washed with 100 L PBS. Thereaction was conducted in low salt buffer (20 mM Tris-HCl pH 7.4, 3 mMMgCl₂, 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, RocheApplied Science) with 1 μL vaccinia infected cell lysate or 1 uL of invitro translated E9 DNA polymerase. The reaction plates were incubatedat 37° C. for 30 min. Total DNA synthesis activities were determinedthrough incorporation of DIG-dUTP using a DIG detection ELISA kit (RocheApplied Science) and its substrate2,2′-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS). The plates wereread at an absorbance of 405 nm on a microplate reader (Tecan GeniusPro, Tecan US).

Plaque Reduction Assay

African green monkey kidney BSC-1 cells were grown in Dulbecco'smodified Eagle medium (DMEM) supplemented with 10% fetal bovine serum(FBS) (Gibco BRL Life Technologies, Gaithersburg, Md.) and 0.1%gentamicin antibiotic at 37° C. in a humidified 5% CO₂ environment.Confluent BSC-1 cells were infected with vaccinia virus at an MOI of0.005 in 48-well plate. One hour post infection, 400 μL of the testcompounds and control were added per well at concentrations ranging from200 nM to 200 μM and incubated at 37° C. for 16 hours. Viridicatumtoxin,tetracycline, and control cidofovir were dissolved in DMSO and dilutedwith the medium. A 5% solution of formaldehyde in PBS was used to fixthe cells. After washing twice with PBS, the plate was stained with 0.2%crystal violet in 50% ethanol.

Cytotoxicity Assay

A cytotoxicity assay that measures the release ofglyceraldehydes-3-phosphate dehydrogenase (GAPDH) was conducted usingthe aCella-TOX bioluminescence cytotoxicity kit (Cell Technology Inc.,Mountainview, Calif.), following the manufacturer's protocol. Briefly,BSC-1 cells were grown to confluency in white 96-well cell cultureplates at 37° C. in DMEM containing 10% FBS and 0.1% gentamicin in thepresence or absence of inhibitor (200 nM to 200 μM). A lysing agent thatproduces maximum release of GAPDH served as a positive control.

Determination of Therapeutic Index

In determining the cellular therapeutic index, no tests were conductedat concentrations greater than 200 μM due to the limited availability ofthe compounds and solubility issues at higher stock concentrations. Theconcentration of inhibitor that causes half of the maximum cellcytotoxicity (CC50) and the concentration that reduces 50% of theplaques (IC₅₀) were used to determine the therapeutics index as follows:

$\left\{ {{{Therapeutic}\mspace{14mu} {Index}\mspace{14mu} ({TI})} = \frac{{Cell}\mspace{14mu} {Cytotoxicity}\mspace{14mu} \left( {CC}_{50} \right)}{{Inhibitory}\mspace{14mu} {Concentration}\mspace{14mu} \left( {IC}_{50} \right)}} \right\}$

Cell Viability Assays

The cell viability at the plaque IC₅₀ value was performed using twoindependent methods, cell counting and MTT assay. For the cell countingmethod, cells were incubated overnight at 37° C. to sub-confluency in48-well plates. The compounds dissolved in DMSO, were further diluted inmedia to a final concentration required to achieve the plaque IC₅₀value. The cells were incubated with the compounds for 24 hrs. The mediawas removed, the cells trypsinized and stained with tryphan blue, andcounted. The MTT assay was also used to confirm cell viability at theplaque IC₅₀. Cells were seeded at 1.5×10⁴ cells/well in a 96-well plateand incubated overnight at 37° C. at 5% CO₂. Compounds dissolved in DMSOwere mixed with media to obtain the concentration required to achievethe plaque IC₅₀ value, and incubated with the cells for 16 h. Each wellreceived 20 μL of the MTT solution (5 mg MTT/mL PBS) and the plate wasrocked for 5 min. The plates were incubated for an additional 5 h tometabolize MTT, after which the media was removed, and the plates wereair dried. To resuspend the formazan, the end product of the MTT assay,200 μL of DMSO was added to each well and the plates were rocked for5-10 min. Absorbance was read at 560 nm.

Quantitative RT-PCR of Vaccinia Genes

BSC-1 cells in 48-well plates were infected with vaccinia virus at anMOI of 30. The test compounds were added to a final concentration of 20μM and incubated at 37° C. Infection time points were obtained byremoving the media, lysing and scraping the cells into pre-cooled tubes.Total RNA from the samples were isolated using RNeasy mini RNA kit fromQiagen and quantified by measuring the absorbance on Nanodrop (NanodropTechnologies, Wilmington Del.). Equal aliquot volumes of each samplewere reverse transcribed according to Superscript first strand DNAsynthesis system (Invitrogen) protocol. Quantitative RT-PCR wasperformed using LightCycler DNA Master Sybr Green from Roche, andprimers designed to probe for early E3, late F9 viral genes and hostGAPDH mRNA expression. The levels of expressed viral genes werenormalized according to the level of GAPDH. The primer pairs used were:F9L Fwd GGACAGTTTAAAAATTGCGCGCTCCG-F9L (SEQ ID No. 3) RevCGTCTAGATCTATTC CTATTT CTTCAG CGATAGC (SEQ ID No. 4) B5R FwdCTTCGGATCCAAATGCTGTCTGCG (SEQ ID No. 5) B5R Rev CGCCGTTGCAACTTAGTGTCATGGTG (SEQ ID No. 6) E3L Fwd GGAATCGAA GGAGCTACTGCTGCAC E3L (SEQ IDNo. 7) Rev CTTATCCGCCTCCGTTG TCATAAACC (SEQ ID No. 8) gapdh FwdCCATGGTGAAGGTGAAGACTGC (SEQ ID No. 9) GAPDH Rev CAGCCTTGAC AGTGC CATGG(SEQ ID No. 10). The thermal cycler conditions were 10 min at 95° C., 45cycles of 5 s at 95° C. followed by 5 s at 60° C. and 5 s at 72° C. Allof the samples were assayed in duplicate. A DNA standard calibrationcurve was plotted using known concentrations of standard cDNA andprimers.

Western Blot Analysis

Cells were harvested and lysed at 4° C. Cells were pelleted (300×g/2min) and 4×LDS was added to the supernatant that was heated to 95° C.and loaded onto a 10% Bis-Tris gels (Invitrogen). Proteins on the gelwere transferred onto a nitrocellulose membrane and probed with primaryE3, L1 and GAPDH antibodies, secondary anti-mouse or anti-rabbitperoxidase conjugated antibodies, and visualized by chemiluminescence(Pierce). The vaccinia antibodies were kindly provided by Drs. G. Cohenand R. Eisenberg.

Example 2 Screening of Chemical Libraries for Compounds that BlockVaccinia Virus DNA Synthesis

Using the invented mechanistic Rapid Plate Assay (See also U.S. Pat. No.6,204,028), a chemical library was screened for compounds that blockvaccinia virus DNA synthesis. Then compounds that blocked vaccinia virusDNA synthesis in the Rapid Plate Assay were further tested forpreventing vaccinia virus from infecting tissue culture cells (plaquereduction) with minimal toxicity.

The following compounds (the details of which are set forth in Table 2)were effective in blocking the replication of vaccinia virus in cellsand thus can be as therapeutic compounds for blocking smallpox virusreplication. Additional particularly effective compounds were found(structures XLIV and XLV).

-   I.    (E)-N1-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-N2,N2-dimethylethane-1,2-diamine.-   II. (E)-2-(6-methyl-2-phenyl-4H-chromen-4-ylideneamino)ethanol-   III. (E)-N-(6-methyl-2-phenyl-4H-chromen-4-ylidene)propan-1-amine-   IV. Flufenamic Acid-   V. Apigenin-   VI. Ro 31-8220; Bisindolylmaleimide IX; or Carbamimidothioic acid,    3-[3-[2,5-dihydro-4-(1-methyl-1H-indol-3-yl)-2,5-dioxo-1H-pyrrol-3-yl]-1H-indol-1-yl]propyl    ester (9CI)-   VII. (2-(2,4-dimethoxyphenyl)indolizin-3-yl)(pyridin-3-yl)methanone-   VIII. (E)-methyl 9-(hydroxyimino)-9H-fluorene-4-carboxylate-   IX. 6H-Indolo[2,3-b]quinoxaline-6-acetic acid, 9-chloro-(9CI)-   X. 6H-Indolo[2,3-b]quinoxaline-6-(2-morpholinoethy)-   XI. 1H-Imidazole,    2-[[(2,4-dichlorophenyl)methyl]thio]-1-(ethylsulfonyl)-4,5-dihydro-(9CI)-   XII. 2-(2,6-dichlorobenzylthio)-6-methylpyrimidin-4-yl    furan-2-carboxylate-   XIII.    (Z)-5-imino-6-((4-oxo-4H-chromen-3-yl)methylene)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7(6H)-one-   XIV. 3-(4-chlorophenylthio)-6-methyl-1H-indole-2-carboxylic acid-   XV. Benzoic acid,    3-[[(3,5-dibromo-2-hydroxyphenyl)methylene]amino]-2-methyl-(9CI)-   XVI. (E)-2,4-dichloro-6-((5-methylisoxazol-3-ylimino)methyl)phenol-   XVII. 4H-Thiazolo[5,4-b]indole, 4-acetyl-2-methyl-(9CI)-   XVIII. Imidazo[2,1-b]benzothiazole-7-carboxamide,    N-(1-ethyl-2-pyrrolidyl)methyl-2-(3-methoxyphenyl)-(9CI)-   XIX.    N-tert-butyl-4-(7-methylthieno[3,2-d]pyrimidin-4-yl)piperazine-1-carboxamide-   XX. CA Registry Number: 874880-06-5-   XXI. “N-Fmoc-anthranilic acid; Benzoic acid,    2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-(9CI)”-   XXII. 4-Quinazolinamine,    N-(2,4-dimethoxyphenyl)-2-(4-methyl-1-piperazinyl)-(9CI)-   XXIII. Benzoic acid,    2-[1-[[amino[(7-methoxy-4-methyl-2-quinazolinyl)imino]methyl]imino]ethyl]-(9CI)-   XXIV.    (E)-N4(9-ethyl-9H-carbazol-2-yl)methylene)-4H-1,2,4-triazol-4-amine-   XXV. CA Registry Number: 902915-02-0-   XXVI. 1,4-Pentadien-3-one, 1,5-di-2-furanyl-(9CI)-   XXVII. Methyl    6-isopropyl-2-(4-methoxyphenylamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate    hydrochloride-   XXVIII.    (E)-4-(3-(ethoxycarbonyl)-5-methyl-4-phenylthiophen-2-ylamino)-4-oxobut-2-enoic    acid-   XXIX. Tipindol; Tipindole; Typindole;    Thiopyrano[4,3-b]indole-8-carboxylic acid, 1,3,4,5-tetrahydro-,    2-(dimethylamino)ethyl ester (7CI,8CI,9CI)-   XXX. 2,6-bis(4-(dimethylamino)phenyl)pyrylium-   XXXI.    (E)-1-ethyl-5-((E)-3-(furan-2-yl)allylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione-   XXXII.    (E)-5-(furan-2-ylmethylene)-2-imino-3-(thiazol-2-yl)thiazolidin-4-one-   XXIII. 1H-Pyrrole-3-carboxylic acid,    1-(4-carboxyphenyl)-2-methyl-5-phenyl-, 3-ethyl ester (9CI)-   XXXIV. 2-(2-chloro-5-(trifluoromethyl)phenylamino)-5-methoxybenzoic    acid-   XXXV.    3-(2-bromobenzyloxy)-6-methyl-1-(thiophen-2-yl)-5,6,7,8-tetrahydro-2,6-naphthyridine-4-carbonitrile-   XXXVI. 2-amino-3-(9-(4-chlorophenyl)-9H-fluoren-9-ylthio)propanoic    acid-   XXXVII. Fentichlor; 2,2′-thiobis(4-chlorophenol)-   XXXVIII. Tetracycline;    4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide    methanesulfonate.-   XXXIX.    (E)-3-(4-chloro-3-nitrobenzylideneamino)-2-thioxothiazolidin-4-one.-   XL. 6,6′-(ethane-1,2-diylbis(oxy))bis(2-methylquinolin-4-amine).-   XLI. 2,2′-sulfonylbis(4-chlorophenol).-   XLII.    4,4′-(2,2′-azanediylbis(ethane-2,1-diyl)bis(tellanediyl))diphenol.

Example 3 Identification of Polymerase and Processivity Inhibitors ofVaccinia DNA Synthesis Using a Stepwise Screening Approach Step I.Primary High-Throughput Screen

For this study, the rapid plate assay was performed in a microtiterplate in which a 100-nucleotide template with biotin covalently linkedto its 5′ end was immobilized onto the streptavidin-coated wells. A20-nucleotide primer was annealed to the 3′ end of template. Vaccinialysate was added to each well of the plate to allow incorporation of thefour dNTPs as well as digoxigenin-dUTP. A peroxidase-conjugatedanti-digoxigenin antibody that recognizes digoxigenin in the newlysynthesized DNA generated a colorimetric reaction that was quantifiedwith a plate reader at 405 nm.

The rapid plate assay was used to screen the NCI Diversity and TrainingSet library of 2,222 compounds. This library contains diversethree-dimensional structures representative of approximately 140,000compounds from the NCI-DTP library. For this primary screen, allcompounds were tested at a concentration 167 μM. The results of atypical plate are represented in FIG. 2. Compounds that inhibit DNAsynthesis (hit compounds) were identified by a decrease in absorbance at405 nm. A total of 169 compounds decreased DNA synthesis by 50% orgreater, giving a hit rate of 7.6%.

Step II. Selectivity Screen

169 hit compounds obtained from the primary screen (Step I) were testedto determine if these vaccinia inhibitors were also able to block DNAsynthesis conducted by a completely different polymerase andprocessivity complex. For this purpose, the unrelated KSHV DNApolymerase (Pol8) and processivity factor (PF8) were used. Compoundsthat block DNA synthesis of vaccinia virus but not KSHV are of greatestinterest since irrelevant reasons for the inhibition (e.g. DNAintercalation and DNA groove binding) are eliminated. Moreover, thesevaccinia virus selective inhibitors unlikely bind to conservedstructural motifs of the KSHV and vaccinia proteins. Compounds in step11 that inhibited KSHV DNA synthesis by 50% or greater were considerednon-specific. The results of this step yielded 47 compounds, giving ahit rate of 2.1% from Step I (2,222 compounds). Results of one of theselectivity plates are presented in FIG. 3. The compounds that inhibitedboth KSHV and vaccinia were set aside for future cytotoxicity testingsince they have a general preference for viral proteins as opposed tocellular proteins.

Step III. Screen to Distinguish Polymerase Vs. Processivity Inhibitors

A20, D4 and E9 proteins are necessary and sufficient for vacciniaprocessive DNA synthesis. Thus the assays were designed to distinguishbetween vaccinia inhibitors that target nucleotide incorporation by E9alone from inhibitors that target processive incorporation of dozens tothousands of nucleotides by the triad, A20, D4 and E9.

To distinguish polymerase from processivity inhibitors, altered versionsof the plate assay were employed. To define polymerase inhibitors, E9activity was examined in low salt. In low salt, DNA polymerases wereable to synthesize fully extended strands in the absence of processivityfactors. The uniform template, which contains evenly distributed sitesfor DIG-dU incorporation, was used to test for polymerase inhibitors(FIG. 4A). By contrast, to identify processivity inhibitors, theexperiments were performed in high salt, in which E9 exhibits limitednucleotide incorporation whereas processive strand synthesis by the A20,D4, E9 triad is preferred. To assay for processivity, the distaltemplate was used to allow DIG-dU to be incorporated only at the distal3′ end of the nascent DNA. Two compounds (69343 and 55636) weredesignated as polymerase inhibitors since they prevented E9 fromincorporating nucleotides on the uniform template (FIG. 4B). Asexpected, these polymerase inhibitors also blocked the distal template(FIG. 4D), since vaccinia DNA synthesis is dependent on theincorporation of dNTPs by E9. In contrast, two compounds (123526 and124808) were designated as processivity inhibitors since they preventedDNA synthesis by the triad on the distal template (FIG. 4D) but not onthe uniform template (FIG. 4B).

Of the 47 compounds that passed Step II, 43 compounds did not inhibiteither the polymerase or processivity assays. This result was due to thedifferent protein sources used—infected cell lysate in Step I vs. invitro translated proteins in Step III. Thus screening stringency can begreatly increased by switching from one protein source to another.

Step IV. M13 DNA Synthesis Assay

The ability of compounds to inhibit DNA synthesis in the rapid plateassay was confirmed using the M13 DNA synthesis assay. In the M13 assay,full-length DNA strands of 7,249 nucleotides are produced and visualizedby autoradiography. Because of the length of the product, the M13 assayserved as a rigorous test for processive DNA synthesis. As shown in FIG.5, lane 2, full-length double stranded M13 DNA was successfullysynthesized in the presence of the A20, D4, E9 triad. The brackets shownin FIG. 5 indicate the formation of greater than unit length products.The synthesis of M13 DNA was blocked by increasing concentrations of thetwo polymerase inhibitors NSC 69343 (lanes 3-7) and NSC 55636 (lanes8-12), and the two processivity inhibitors NSC 123526 (lanes 13-17) andNSC 124808 (lanes 18-22). Most notably, the polymerase inhibitor NSC69343 completely inhibited M13 DNA synthesis at 100 μM (lane 5). All ofthe other compounds inhibited at concentration between 100 μM and 1 mM.

Step V. Plaque Reduction Assay

A plaque reduction assay was used to evaluate the antiviral activity ofthe two polymerase inhibitors and the two processivity inhibitors thatwere selected from the screening assays (FIG. 6). BSC-1 cells wereinfected with 100-150 pfu of vaccinia virus and the inhibitors wereadded to the cell monolayer 1 h post-infection at a final concentrationof 100 μM. This final concentration of 100 μM was based on the abilityof known inhibitors to reduce plaque formation by approximately 50%following infection of BSC-1 cells by vaccinia virus. The results aresummarized in Table I. Surprisingly, NSC 69343, the more potentpolymerase inhibitor based on in vitro assays, was less remarkable inits ability to reduce viral plaques (40%), as compared to NSC 55636, theless potent polymerase inhibitor, which caused significant plaquereduction (95%). As for the two processivity inhibitors, NSC 123526showed appreciable plaque reduction (72%) as compared with NSC 124808(36%).

Step VI. Cytotoxicity Assay

Cytotoxicity of the four inhibitors of vaccinia DNA synthesis wasdetermined using the aCella-Tox assay. This assay quantifies thecellular release of glyceraldehyde-3-phosphate dehydrogenase (G3PDH)which is essential for the production of ATP in the glycolysis pathway.G3PDH released into the cell media is used in a coupled reaction togenerate ATP, which is then detected by luciferase/luciferinbioluminescence. As indicated in Table I and FIG. 6, at 100 μM, all ofthe inhibitors resulted in cell cytotoxicity lower than 50%. The twoprocessivity inhibitors (NSC 123526 and 124808) exhibited the samelevels of cytotoxicity (21%) whereas the polymerase inhibitors (NSC55636 and 69343) exhibited toxicities of 14% and 17% respectively. Mostinterestingly, the least cytotoxic inhibitor, NSC 55636, is also themost potent plaque reducer.

Cellular Therapeutic Indices for Polymerase Inhibitor NSC 55636 andProcessivity Inhibitor NSC 123526

The polymerase inhibitor NSC 55636 and processivity inhibitor NSC 123526were considered to be the most significant compounds based on theirefficacy at reducing vaccinia virus plaques with the least cytotoxicity.To establish the cellular therapeutic index

$\left\{ {{{Therapeutic}\mspace{14mu} {Index}\mspace{14mu} ({TI})} = \frac{{Cell}\mspace{14mu} {Cytotoxicity}\mspace{14mu} \left( {CC}_{50} \right)}{{Effective}\mspace{14mu} {Concentration}\mspace{14mu} \left( {EC}_{50} \right)}} \right\}$

for each compound, plaque reduction and cell cytotoxicity at a range ofconcentrations between 100 nM-200 μM was measured (Table 2). NSC 55636gave a plaque reduction EC₅₀ of 5 μM and CC50 of greater than 200 μM,resulting in a cellular TI of greater than 40. NSC 123526 gave a plaquereduction EC₅₀ of 65 μM and CC50 of greater than 200 μM, resulting in acellular TI of greater than 3.

These experimental settings provided: the selection of functionalinhibitors that block vaccinia DNA synthesis; elimination of general andirrelevant inhibitors; distinguish whether the polymerase or theprocessive mechanism is targeted. The first screening step, which usesinfected cell lysate, was designed for poxviruses, which are uniqueamongst the DNA viruses in that they replicate in the cytoplasm. Thisprovides a great advantage since there are no competing nuclearpolymerases in the cytoplasmic lysate that contribute to anomalousresults. The second screening step eliminates irrelevant inhibitors suchas DNA intercalators and DNA groove binders. Moreover, this stepidentifies antiviral compounds that were directed against viralpolymerases. The third screening step, which distinguishes inhibition ofthe polymerase or processive mechanism, is also applicable to otherviruses and eukaryotes that engage processivity complexes in their DNAsynthesis. Using this stepwise approach, promising therapeutics thattarget the processivity complex of poxviruses were successfully found.

The stepwise screening delivered two compounds which have distinct modesof inhibiting polymerase/processive vaccinia DNA synthesis. The firstcompound, NSC 55636, inhibited the catalytic activity of E9 DNApolymerase (Tables 1 and 2). By contrast, the second compound, NSC123526, inhibited the processive activity of the triad A20, D4 and E9(Tables 1 and 2).

The polymerase inhibitor NSC 55636, also known as Fentichlor, is anantibacterial, anthelmintic and antifungal agent. This study shows thatNSC 55636 is a promising poxvirus inhibitor due to its high cellulartherapeutic index of greater than forty, reflecting its ability toeffectively block vaccinia virus infection with minimal cytotoxicity.

The processivity inhibitor NSC 123526, is an S-fluorenylcysteinecompound. The cysteine moiety renders the compound cell permeable anddelivers the flourenyl group to the interaction site, inducing localconformational changes.

This study has yielded two other compounds that were effective inblocking vaccinia DNA synthesis in the in vitro rapid plate assays,while not as effective in cell-based assays. The polymerase inhibitorNSC 69343 is in fact tetracycline, an antibiotic that inhibits theprokaryotic 30S ribosome (Tables 1 and 2).

In summary, the sequential rapid plate screening provides a clear routefor the identification of lead compounds with inhibitory activitiestowards poxvirus DNA synthesis. The stepwise design incorporated filtersthat (i) examined compounds that block viral DNA synthesis, (ii)selectively removed compounds that inhibited DNA synthesis in a generalor irrelevant manner, and (iii) distinguish whether the inhibitors wereblocking polymerase or processive mechanisms. This screening strategyyielded two candidate compounds for vaccinia, NSC 55636 and NSC 123526,representing a hit rate of 0.1%. Characterization of the A20, D4, E9triad will provide insights into the mechanism of inhibition by thesecompounds and allow us to generate analogs that achieve maximumantiviral activity. The sequential screening approach can be modified toidentify polymerase and processivity inhibitors of other viruses andmicrobial agents.

Design and Optimization of the Rapid Plate Assay for High-ThroughputScreen

Infected cytoplasmic extracts were used as the source of viral proteinsfor the rapid plate assay. Poxviruses are unique amongst DNA viruses inthat their replication occurs solely within the cell cytoplasm. Thisprovided an advantage for preparing viral replication proteins that werefree of nuclear enzymes involved in cellular DNA synthesis. Cytoplasmicextracts, prepared from vaccinia-infected BSC-1 cells, were shown to becompletely separated from nuclear proteins, as verified by Western blotwith the cytoplasmic marker β-Actin and the nuclear marker Rb1 (FIG.7A). Moreover, cytoplasmic extracts of uninfected cells had little to noDNA synthesis activity, further indicating that the DNA synthesisactivity of infected cell extracts was exclusively due to vacciniacytoplasmic components and not to nuclear contaminants (FIG. 7B). Beforeproceeding with the major high-throughput screening (HTS), the assay wasadapted to a 384-well platform. DNA synthesis reactions were performedin streptavidin-coated, 384-well plates for 30 minutes at 37 C, in afinal reaction volume of 30 μL. Each well contained 100 mM ammoniumsulfate, 0.5 μM digoxigenin-11-2′-2 deoxy-uridine-5′-triphosphate, 2pmoles biotinylated primer-template and 0.2 mU vaccinia DNA polymerase(see Methods for details). The assay was evaluated for plate uniformityand signal variability by testing three plates on two different days. Nodrift or edge effects were observed and the calculated values forsignal-to-background, signal-to-noise, coefficient of variation, andscreening window coefficient (Z′-factor) validated the assay as suitablefor HTS. The results were reproducible and the screening windowcoefficient (Z′-factor) had an excellent value of 0.95. Next, the assaywas validated by robotically testing 1,520 chemical compounds with knownpharmacological activities. Out of the 58 hit compounds that blocked thecolorimetric reaction, 39 are known to bind DNA or inhibit DNA and RNApolymerases, which indicated that the assay is capable of detectinginhibitors of DNA synthesis. A survey of the current literature foundthat the remaining 19 hit compounds were never reported as inhibitors ofDNA synthesis.

High-Throughput Screening for Inhibitors of Vaccinia-Dependent DNASynthesis

The rapid plate assay was used to perform a robotic HTS to evaluate atotal of 45,832 low-molecular-weight synthetic compounds (MW<500) and2,311 natural extracts, that had been partially purified from endophyticfungi or plants used in traditional Chinese medicine (Table 3). Allsynthetic compounds and natural extracts, obtained as lyophilizedsolids, were dissolved in DMSO. The synthetic compounds from 11independent libraries (Table 3) were each tested in duplicate, at aconcentration of 16.7 μg/mL, equivalent to a molar concentration of33-167 μM, depending on the molecular weight of the actual compound. Thenatural extracts, resuspended in DMSO at 15 mg/mL, were also tested induplicate at 50 μg/mL final working concentration. From screening atotal of 49,663 synthetic compounds and natural extracts, 829 inhibitorsthat block vaccinia virus DNA synthesis were identified, whichrepresents a total hit rate of 1.6%. Inhibitors were classified as weak(30-50% inhibition), medium (50-70% inhibition), and strong (greaterthan 70% inhibition), based on the colorimetric intensity of the readoutsignal relative to the control signal obtained with no inhibitor (DMSOalone). Of the total number of 829 identified inhibitors, 178 werestrong, 271 were medium, and 380 were weak inhibitors (Table 3).Interestingly, the percentage of natural extracts that inhibited DNAsynthesis (hit rate) was much greater than that obtained with thesynthetic compounds (Table 3). Likely, the DNA synthesis reaction ismuch more vulnerable to the plethora of chemical entities present innatural extracts than the singly purified compounds from syntheticlibraries. Of note, the group of synthetic compounds with knownbiological activities had a greater hit rate than the syntheticcompounds from combinatorial libraries, which is not surprising sincemany of these known compounds carry out their biological activities bymodulating processes that involve DNA. 145 of the synthetic compoundsthat represented all of the strong and most of the intermediateinhibitors from the combinatorial synthetic libraries were retested. Ofthese 145 compounds, 135 were confirmed to block vaccinia DNA synthesisupon manual retesting in the rapid plate assay using 96-well plates. The135 confirmed hit compounds were classified into 59 different chemicalfamilies based on their molecular structure. From these syntheticcompounds, 93 strong and intermediate inhibitors that cover allstructural families of the hit compounds were purchased and evaluated infollow-up studies. The natural extracts were not further pursued in thisstudy, since they need to be fractionated in order to purify andidentify the active components.

Dose-Dependent Inhibition of Vaccinia-Catalyzed DNA Synthesis andDetermination of the IC₅₀ Values in the Rapid Plate Assay

The 93 synthetic compounds (above) were analyzed in both enzymatic andin vitro cell-based assays. Compounds were suspended in DMSO at a finalconcentration of 20 mM and tested manually in the rapid plate assay forinhibition of DNA synthesis catalyzed by vaccinia proteins.

The assay was performed in 96-well plates in conditions similar to thoseused for the primary high-throughput screen. Compounds were tested intriplicate over a range of concentrations and the percentage inhibitionvalues were fitted to sigmoidal dose-response curves. For each compounda dose-dependent inhibition curve was generated, from which the 50%inhibitory concentration (IC50) was determined. The dose-response curvesfor five of the compounds is presented in FIG. 8. All chemicalsinhibited vaccinia virus catalyzed DNA synthesis in the rapid plateassay. The IC50 values for 89 compounds ranged from 0.5 μM to 400 μM,while 4 compounds showed only partial inhibition at concentrations of400 μM or higher.

Testing the DNA Synthesis Inhibitors for their Ability to Block VacciniaVirus Infection In Vitro

The 93 DNA synthesis inhibitors (above) were first tested in a plaquereduction assay to stop the formation of vaccinia virus plaques on BSC-1cell monolayers. The plaque reduction assay uses a very low amount ofvaccinia virus, just enough to give approximately 50 plaques in eachwell of a 48-well plate. Each compound was tested in triplicate and theresults were used to estimate the compound concentrations for which thenumber of viral plaques was reduced to approximately half (effectiveconcentration 50 or EC50) (Table 4). Next, the 93 compounds were testedin a cell protection assay. The cell protection assay was performed withamounts of virus capable of lysing every cell in the culture during the20 hour incubation period. Compounds with antiviral properties preservethe monolayer's integrity and the adherent cellular mass is stained togenerate the read-out signal (absorbance at 570 nm). Each compound wastested in triplicate over a range of concentrations up to 200 μM intwo-fold serial increments and dose-dependent plots were generated byfitting the results on four-parameter sigmoidal response curves. Theseplots were used to calculate the compound concentrations at which 50%antiviral protection was observed, as compared to the unprotected DMSOcontrol. While both these assays measure the antiviral activity, theydiffer by the amount of virus used to infect the cell monolayer. As itcan be seen in Table 4, there is an overall concordance between the EC50values obtained in the plaque reduction assay and the cell protectionassay, which suggests that both assays are good indicators of acompound's efficacy to block viral infection.

Cytotoxicity of the Hit Compounds

The cell protection assay (above) indirectly measures the compounds'cytotoxicity, in that compounds which successfully protect against viralinfection must do so without destroying the cells. Furthermore, thecompounds' cytotoxicity was assessed in an assay that measures themetabolic activity of cells exposed to chemicals in the absence ofvirus. After culturing the cells in the presence of compounds, cellviability was determined by adding a cell-impermeable water-solubletetrazolium salt that is reduced extracellularly to a dark-red solubleformazan and is monitored spectrophotometrically. Cytotoxicity wasdetermined by measuring the reduction of tetrazolium salt due tometabolic activity of BSC-1 cells exposed to compounds, relative to DMSOalone. The percent cytotoxicity was fitted onto four-parameter sigmoidaldose response curves and the 50% toxic concentration values (TC50) weredetermined. As expected, cytotoxicity increases with the incubationtime. Some compounds exhibited very little cytotoxicity in overnightcultures and their cytotoxic effects for 3 or 5-day incubation periodwere determined.

Compound Trapping by Extracellular DNA

The compounds identified using HTS were selected based on their abilityto block vaccinia-catalyzed DNA synthesis irrespective of the mechanismof inhibition. Some of these inhibitors function by binding DNA and thusstopping DNA strand extension by polymerase. Such compounds affectindiscriminately many cellular and viral processes that involve DNA. Toinvestigate if the hit compounds have affinity for DNA, antiviralactivity was tested in the presence of large amounts of genomicdouble-stranded DNA added in the cell/virus culture. Since cells do nottake up DNA under normal culturing conditions, this exogenous DNAremains outside the cells and traps any DNA-binding compound, resultingin (which leads to) a loss of that compound's antiviral activity.

Calculation of the Selectivity Index

The selectivity index for each compound was calculated as the ratiobetween the TC50 in the 20 hour cytotoxicty assay and the EC50 in thecell protection assay. Some compounds with very low toxicity had lessthan 50% cytotoxic effect at the highest tested concentration of 200 μMand their TC50's were estimated as greater than 200 μM in the 20 hoursassay. It is noted that in order to obtain EC50 and TC50 values that canbe compared to generate accurate SI's, the cell protection assay and thecytotoxicity assay need to be performed in similar conditions:fast-growing BSC-1 cells exposed to compounds or virus/compound mixturesfor the same length of time.

Example 4 Inhibition of Vaccinia DNA Synthesis by Viridicatumtoxin

Viridicatumtoxin inhibits vaccinia DNA synthesis in vitro

Viridicatumtoxin and tetracycline were compared for their effectivenessin blocking DNA synthesis using the rapid plate assay. In this assay,DNA synthesis initiated from a primer annealed to a 100-nucleotidetemplate. The primer-annealed template was tethered to astreptavidin-coated plate by a biotinylated moiety on its 5′ end. DNAsynthesis was conducted in low salt which enables vaccinia E9 DNApolymerase, produced by in vitro translation to perform extended strandsynthesis. A failure to incorporate dNTPs in the presence of testcompounds, i.e. viridicatumtoxin or tetracycline, indicates that E9polymerase is inhibited. As seen in FIG. 10 and Table 5, the IC₅₀ valuesobtained for the inhibition of vaccinia DNA synthesis by tetracycline(54 μM) and viridicatumtoxin (63 μM) were similar.

Viridicatumtoxin Blocks Vaccinia Virus Infection

In order to evaluate the ability of viridicatumtoxin to inhibit vacciniavirus infection, a plaque reduction assay was employed. As representedin FIG. 11 and Table 5, viridicatumtoxin was able to reduce the numberof plaques at near nanomolar concentrations (IC₅₀ of 1.6 μM). It isnoted that at very high concentrations of viridicatumtoxin (≧100 μM),the cells began to detach. By contrast, tetracycline was ineffective atreducing vaccinia plaques (IC₅₀>200 ┌M).

Viridicatumtoxin Exhibits Low Cellular Cytotoxicity

To determine the cytotoxicity of viridicatumtoxin on cells, theaCella-TOX assay was used. This assay quantifies the cellular release ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) which is essential forthe production of ATP in the glycolysis pathway. Briefly, GAPDH that isreleased into the cell media is used in a coupled reaction to generateATP, which can be detected by luciferase/luciferin bioluminescence. TheCC₅₀ for viridicatumtoxin in the GAPDH release assay was greater than200 μM (Table 5), indicative of low cellular cytotoxicity. Thetherapeutic index value for viridicatumtoxin, calculated from the cellcytotoxicity CC₅₀ and plaque reduction IC50, was greater than 125.

Viridicatumtoxin does not Affect Cell Viability at the Plaque IC₅₀

Two different assays were used to determine the viability of the cellsat the IC₅₀ concentration of viridicatumtoxin (1.6 μM). About 90% of thecells remained viable as determined by the cell count assay, and about85% of the cells remained viable as measured by the MTT assay (Table 5).

Viridicatumtoxin Prevents Vaccinia Virus Late Gene Expression

Late viral gene expression is dependent upon vaccinia DNA synthesis. Totest if viridicatumtoxin prevents expression of vaccinia late genes, butnot early genes, representative viral marker genes were used to evaluateearly and late vaccinia expression in the presence and absenceviridicatumtoxin. A quantitative RT-PCR analysis to detect early andlate viral mRNA production was performed first. RNAs from different timepoints of viridicatumtoxin-treated (20 μM) and untreated vaccinia virusinfected cells were converted into cDNAs using oligo-dT. Specific primerpairs were used to amplify regions of the vaccinia early E3 vaccinialate F9 and cellular GAPDH cDNAs by quantitative real time PCR. Forquantitation, GAPDH was used to normalize the levels of E3 and F9. Asshown in FIG. 12A, both in the presence and absence of viridicatumtoxin,production of early E3 mRNA was maxiumum at 3 h.p.i. and dropped back tolow levels by 5 h.p.i. By contrast, as shown in FIG. 12B, in the absenceof viridicatumtoxin, the production of late F9 mRNA began to increase at5 h.p.i. and continued to increase thereafter. However, in the presenceof viridicatumtoxin, there was only a marginal increase in F9 mRNAexpression by 5 h.p.i. that plateaued until the last time point (8h.p.i.). The prevention of vaccinia late mRNA expression byviridicatumtoxin was substantiated by examining vaccinia early and latemarker proteins by western blot analysis. As shown in FIG. 13, inuntreated infected cells, the E3 marker protein appeared at 2 hpost-infection, and continued to increase to the last time point (8h.p.i.). In the presence of viridicatumtoxin, there was only a slightdelay in appearance of E3 (3 h.p.i.). However, there is no significantdifference in the levels of E3 in the presence or absence ofviridicatumtoxin at 5 and 8 h.p.i. By contrast, the vaccinia late markerprotein L1 which is involved in viral assembly (26, 27) (31), wassignificantly decreased in the presence of 20 μM viridicatumtoxin bothat the time of its appearance at 5 h.p.i. as well as at 8 h.p.i. Thisability of viridicatumtoxin to block late viral protein expression is inaccord with its ability to inhibit late mRNA expression.

TABLE 1 Selected inhibitors of the invention Targeted Inhibition NSCActivity of Cell Inhibitor Structure (% Inhibition) plaques*Cytotoxicity* 123526

Processivity (64%) 72% 21% 124808

Processivity (62%) 36% 21%  55636

Polymerase (74%) 95% 14%  69343

Polymerase (92%) 40% 17%

*All compounds were tested at a fixed concentration of 100 μM. *Allcompounds were tested at a fixed concentration of 100 μM.

TABLE 2 The efficiency of NSC 55636 and NSC 123526 as vacciniainhibitors Inhibitor compound CC50^(a) EC₅₀ ^(b) TI^(c) NSC 55636 >200μM  5 μM >40 NSC 123526 >200 μM 65 μM >3 Table 2. ^(a)Concentration atwhich compound is 50% cytotoxic ^(b)Concentration at which compoundreduced plaques by 50% ^(c)Ratio of CC50 to EC₅₀

TABLE 3 Hit rate Number of Intermediate Compounds in Strong InhibitionInhibition Weak Inhibition Library Type Library Name Library >70%Inhibition 50%-70% Inhibition 30%-50% Inhibition Known Bioactives BiomolICCB Known Bioactives 480 6 (1.3%) 8 (1.7%) 11 (2.3%) NINDS CustomCollection 1,040 15 (1.4%) 8 (0.8%) 10 (1.0%) Natural Extracts StarrFoundation Extracts 2 1,000 43 (4.3%) 41 (4.1%) 57 (5.7%) ICBG FungalExtracts 1 851 59 (6.9%) 57 (6.7%) 54 (6.3%) ICBG Fungal Extracts 2 46018 (3.9%) 30 (6.5%) 24 (5.2%) Synthetic ChemDiv 3 16,544 21 (0.1%) 64(0.4%) 89 (0.5%) Compounds MixCommercial 5 268 0 (0.0%) 0 (0.0%) 1(0.4%) Maybridge 4 4,576 2 (<0.1%) 9 (0.2%) 28 (0.6%) ActiMol TimTec 18,518 6 (0.1%) 19 (0.2%) 25 (0.3%) Bionet 2 1,700 2 (0.1%) 2 (0.1%) 14(0.8%) Enamine 1 6,004 1 (<0.1%) 9 (0.1%) 19 (0.3%) I.F. Lab 1 6,543 4(0.1%) 17 (0.3%) 40 (0.6%) I.F. Lab 2 292 0 (0.0%) 4 (1.4%) 0 (0.0%)Maybridge 2 704 1 (0.1%) 1 (0.1%) 5 (0.7%) MixCommercial 4 331 0 (0.0%)1 (0.3%) 3 (0.9%) Peakdale 2 352 0 (0.0%) 1 (0.3%) 0 (0.0%) Total 49,663178 (Hit Rate 0.4%) 271 (Hit Rate 0.5%) 380 (Hit Rate 0.8%)

TABLE 4 Summary of activities DNA Synthesis Cell Protection PlaqueCytotoxicity Cytotoxicity Cytotoxicity Selectivity Inhibition 20 hReduction 20 h 20 h 72 h 120 h Index Effect of Compound IC50 (uM) EC50(uM) EC50 (uM) CC50 (uM) CC50 (uM) CC50 (uM) SI dsDNA 1304-M19 42 23 25100 4.3 Yes 1306-M02 56 12 6 23 1.9 Yes 1394-F14 131 50 50 >200 363 >4.0 No 1398-K11 25 87 100 >200 152 60 >2.3 Yes 1410-N05 5.6 25 10 200.8 Yes 1417-H08 30 40 25 87 2.2 Yes 1421-I18 374 30 25 159 5.3 No1427-G18 108 9.6 10 127 20 13.2 No 1429-L06 46 100 50 >100 13 30 >1 No1430-M18 188 90 25 166 25 1.8 No 1488-G10 25 14 25 133 9.5 Yes 1488-N05708 193 100 >200 35 >1 Yes 1489-L17 25 57 40 >200 12 >3.5 Yes 1489-N1718 57 25 >200 10 >3.5 Yes 1492-H05 65 18 40 192 10.7 Yes 1501-M18 39 98100 135 1.4 Yes 1502-N02 75 188 150 >200 >200 50 >1 No 1502-O21 46 41 25143 3.5 Yes 1504-I21 43 130 75 >200 174 120 >1.5 No 1508-M17 26 10075 >200 38 20 >2 No 1513-N12 111 236 100 >200 >200 75 >1 No 1522-I19 2523 10 29 1.3 Yes 1523-L14 12 50 15 157 40 3.1 No 1526-I08 26 104 75 >20030-50 20 >1.9 No 1526-P05 129 45 15 115 5 2.6 No 1530-I10 52 100 75 >200Pp at 100 >2 No 1531-O02 30 95 75 >200 ~50 20 >2.1 No 1537-L02 32 8075 >100 >50 >1.2 No 1538-E21 239 100 50 >100 >1 No Cidofovir 13250 >2.500 >500 300 >18.9 No Ara-C 0.24 0.5 16 66.7 Yes Ethidium 100 YesQuinacrine 17 Yes Aphidicolin 18 No

TABLE 5 Summary of viridicatumtoxin inhibition of vaccinia virus DNAsynthesis and infection. % cell viability Plaque on plaque IC₅₀ E9activity IC₅₀ Cell IC₅₀ (μM) (μM) CC₅₀ TI count MTT Viridicatumtoxin 63± 2.8 1.6 ± >200 >125 92 ± 23 83 ± 16 2.4 Tetracycline 54 ±2.1 >200 >200 ND ND ND

Example 5 Identification of Non-Nucleoside DNA Synthesis Inhibitors ofVaccinia Virus by High Throughput Screening Experimental Details PrimaryHigh-Throughput Screen

For HTS, the rapid plate assay was adapted to a robotic platform. TheDNA synthesis reactions were performed in 384-well plates coated withstreptavidin (SigmaScreen plates, Sigma-Aldrich cat. no. S8686). In eachwell, reactions were conducted with 0.5 μL of vaccinia virus to extractcontaining 0.2 mU DNA polymerase activity in 20 mM Tris-Cl pH7.5, 100 mMammonium sulfate, 5 mM MgCl₂, 0.1 mM EDTA, 0.5 mM DTT, 4% glycerol, 40μg/mL BS A, 5 μM each of dATP, dCTP, and dGTP, 4 μM dTTP, 0.5 μMDIG-11-dUTP, in a total volume of 30 μL. Two pmol of biotinylatedprimer-template dissolved in 30 uL PBS were immobilized onstreptavidin-coated wells. The unbound primer template was removed, andthe wells were washed twice with 50 uL of PBS and loaded with 20 uLreaction buffer (20 mM Tris-Cl pH7.5). Then 100 mL of each compound ornatural extract dissolved in DMSO were transferred from the libraryplates by an Epson transfer robot (Epson Robots, Carson, Calif.) fittedwith a 384-pin array. DNA synthesis was initiated by loading 10 uL of a3× reaction mixture. After brief centrifugation, plates were incubatedat 37° C. for 30 mM The DNA synthesis reaction was stopped with 30 uL ofa solution containing 50 mM EDTA and 2% SDS in 10 mM Tris pH 8.Incorporation of DIG-11-dUTP in the newly synthesized DNA strand wasdetected with peroxidase-conjugated antidigoxigenin antibody(anti-DIG-POD). Wells were flow-washed with 400 uL of PBS containing0.1% Tween-20 at the lowest dispensing speed and loaded with 4.5 mU ofanti-DIG-POD in 30 μL of PBS/blocking solution. After gently rocking atroom temperature for 1 h, the antibody solution was removed and wellswere flow-washed as above. Then 30 uL of2,2′-azino-bis(3-ethylbenzthiazoline)-sulfonate (ABTS) peroxi-dasesubstrate dissolved in citrate buffer was added and plates were gentlyrocked at room temperature for 1 h. Color development was stopped with1.0 uL of 4% SDS and absorbance at 405 nm was measured. Readings fromeach well were divided by the plate median, and the percent inhibitionwas determined relative to the values of uninhibited reaction controls.

Cytoplasmic Extracts of Vaccinia-Infected BS-C-1 Cells

Confluent BS-C-1 cells, infected with vaccinia virus WR at an MOI of 15in the presence of 10 mM hydroxyurea, were harvested 6 h post infection.Hydroxyurea inhibits viral DNA replication and transcription ofintermediate and late viral mRNA's, allowing only the expression ofearly gene products and not the abundant late viral genes. ProcessiveDNA synthesis is an early activity of the vaccinia virus and thus is notaffected by hydroxyurea. Infected cells were carefully scraped off theplates, washed once with hypotonic buffer (1.5 mM MgCl₂, 10 mM KCl, 10mM Hepes pH 7.5), resuspended in cold hypotonic buffer, incubated on icefor 10 min, and lysed by gentle dounce homogenization. Nuclei wereremoved by centrifugation at 1000 g for 10 min. The cytoplasmic extractswere filtered twice through 0.2 μm and shown to be free of infectiousparticles as determined by plaque assay. Glycerol was added to 20% finalconcentration, and aliquots were stored at −80° C.

Screened Libraries

The 1520 chemical compounds with known biological activities used forassay validation were from two collections: BIOMOL ICCB Known Bioactives1 (480 compounds) and NINDS Custom Collection (1040 compounds). A totalof 45832 synthetic compounds, representing 11 independent libraries(ChemDiv 3 (16544 compounds), MixCommercial 5 (268 compounds), Maybridge4 (4576 compounds), ActiMol TimTec 1 (8518 compounds), Bionet 2 (1700compounds), Enamine 1 (6004 compounds), I.F. Laboratory 1 (6543compounds), I.F. Laboratory 2 (292 compounds), Maybridge 2 (704compounds), MixCommercial 4 (331 compounds), and Peakdale 2 (352compounds)) were tested in duplicate at a single concentration of 16.7μg/mL, which is equivalent to a molar concentration of 33-167 μM,depending on the molecular weight of each individual compound. Naturalextracts with inhibitory activity were identified by screening the Stan'Foundation Extracts 2 library (1000 extracts from plants used intraditional Chinese medicine), and two collections of partially purifiedextracts from endophytic fungi: 1CBG Fungal Extracts 1 (851 extracts)and ICBG Fungal Extracts 2 (460 extracts). The natural extracts,resuspended in DMSO at 15 mg/mL, were also tested in duplicate at 50μg/mL final working concentration. The chemical libraries were suppliedby the National Screening Laboratory for the Regional Centers ofExcellence in Biodefense and Emerging Infectious Disease (NSRB) at theHarvard Institute of Chemistry and Cell Biology (ICCB, Harvard MedicalSchool), where the robotic HTS was performed.

DNA Synthesis Assay

Milligram quantities of the HTS hit compounds for follow-up studies werepurchased from various vendors and suspended in DMSO at a finalconcentration of 20 mM. Each hit compound was retested over a range ofconcentrations for its ability to inhibit vaccinia virus DNA synthesis.Assays were performed in 96-well plates in conditions similar to thosefor the HTS. Each compound was tested in triplicate in 2-fold serialdilutions, and the IC50 was calculated with the Prism software (GraphPadSoftware, Inc.) for linear regression.

Viral Plaque Reduction Assay

BS-C-1 cells were maintained in Dulbecco's modified Eagle medium (DMEM)to containing 10% fetal bovine serum (FBS), 50 mg/L gentamicin sulfate(Gibco BRL Life Technologies, Gaithersburg, Md.), and grown at 37° C. ina humidified atmosphere with 5% CO2. For the plaque reduction assay,6×10⁴ cells/well were seeded in 48-well plates and allowed to attachovernight. Cells were infected with 50 plaque forming units (pfu)/wellof vaccinia virus WR in the presence of the HTS hit compounds at variousconcentrations. After 20 h, cells were fixed with 5% formaldehyde in PBSand stained with 0.2% crystal violet dissolved in 50% ethanol. Wellswere washed, dried, and plaques were counted under the microscope. TheEC50 values were estimated as the concentration of compound that causeda reduction in the number of plaques by 50%.

Viral Cell Protection Assay

The cell protection assay was performed using similar conditions to theplaque reduction assay. BS-C-1 cells were seeded in 96-well plates at1.5×10⁴ cells/well and grown overnight. The cells were infected with2000 pfu/well of vaccinia virus WR in the presence of inhibitors. Afterincubating at 37° C. for various times (20, 48, and 72 h), cells werefixed with formaldehyde and stained with crystal violet. The excess dyewas washed away, and the cellular mass fixed on the bottom of the wellswas allowed to dry overnight. Absorbance at 570 nm was measured. Eachcompound was tested in triplicate in 2-fold serial dilutions, and EC50values were calculated using the Prism software for linear regression.

DNA Trap Assay

To investigate if the hit compounds retain their antiviral potency inthe presence of exogenous DNA, the cell protection assay was performedwith 400 μg/mL of type III DNA, sodium salt, from salmon testes (Sigma,Saint Louis, Mo.) added in the growth medium. The final concentration ofcompound-derived DMSO was 1% (v/v) in all cell-based assays. For thecontrol wells that contained no compound, an appropriate volume of DMSOwas added to achieve a final concentration of 1%.

Cytotoxicity Assay

Compound cytotoxicity was measured with the Cell Proliferation ReagentWST-1 (Roche Applied Science, Indianapolis, Ind.) following themanufacturer's protocol. BS-C-1 cells were seeded in 96-well plates at5000 cells/well in 50 /μL growth medium without phenol red. Next day, 50μL/well of growth medium containing the tested compounds were added andcultures were grown for 20 h at 37° C. Then 10 μL of WST-1 reagent wereadded to each well, plates were incubated at 37° C. for 30 min, andabsorbance at 450 nm was measured. Each to compound was tested intriplicate in 2-fold serial dilutions, and TC50 values were calculatedusing the Prism software for linear regression. To measure the compoundcytotoxicity over longer incubation periods, the number of BS-C-1 cellsseeded was adjusted: 1000 cells/well for 3-day cytotoxicity and 200cells/well for 5-day cytotoxicity. This ensured that cells did not growpast confluency by the end of the experiment and that the read-outsignal remained in the linear range.

Results Identification of Small Chemical Inhibitors of Vaccinia VirusDNA Synthesis with a Rapid Plate Assay Designed for High-ThroughputScreening

Inventor of the instant application has developed a rapid plate assay toidentify inhibitors of vaccinia virus DNA synthesis that target theviral DNA polymerase and its associated factors required forprocessivity. Briefly, a 5′-biotinylated DNA template annealed to anoligonucleotide primer at its 3′-end is immobilized ontostreptavidin-coated wells. In the presence of the viral polymerase andits associated factors, dNTP's and digoxigenin-labeled2′-deoxy-uridine-5′-triphos-phate (DIG-11-dUTP) are incorporated intothe newly synthesized DNA strand. DNA synthesis activity is measured bythe detection of digoxigenin using an enzyme-linked antibody thatgenerates a colorimetric reaction.

Replication of poxviruses occurs solely within the cytoplasm.Cytoplasmic extracts from infected BS-C-1 cells were used as the sourceof vaccinia proteins for the rapid plate assay. These cytoplasmicextracts were confirmed to be completely free of nuclear proteins byWestern blot with antibody specific for the nuclear markerretinoblastoma protein Rb1 (FIG. 7A). Moreover, cytoplasmic extracts ofuninfected cells had little to no DNA synthesis activity in the rapidplate assay, indicating that the DNA synthesis activity of infected cellextracts was exclusively due to vaccinia cytoplasmic proteins and not tonuclear enzymes involved in cellular DNA synthesis (FIG. 7B).

Before proceeding with the HTS, the rapid plate assay was adapted foruse with the Epson compound transfer robot (Epson Robots, Carson,Calif.). DNA synthesis reactions were optimized without inhibitors instreptavidin-coated 384-well plates. This robotic procedure exhibitedplate uniformity and no signal variability when tested in multipleplates on different days. No drift or edge effects were observed, andthe calculated values for signal-to-background, signal-to-noise,coefficient of variation, and screening window coefficient (Z′-factor)validated the assay as suitable for HTS. The robotic plate assay wasreproducible, and to the Z′-factor had an excellent value of 0.95.

Next, the optimized assay was validated by testing 1520 chemicalcompounds with known pharmacological activities. These known bioactivecompounds are compiled in two libraries and include more than half ofthe drugs currently approved by the Food and Drug Administration. Out ofthe 58 compounds that blocked the colorimetric reaction of the rapidplate assay, 39 are known to bind DNA or inhibit DNA or RNA polymerases,which confirmed that the assay was indeed capable of detectinginhibitors of DNA synthesis. Interestingly, with respect to theremaining 19 known compounds, this is the first report to reveal thatthey are capable of inhibiting DNA synthesis.

The optimized robotic plate assay was then used to identify novelvaccinia DNA synthesis inhibitors by performing a HTS of 45832 smallsynthetic compounds (MW<500) and 2311 partially purified naturalextracts. In this primary screen, the inventor identified 383 naturalextracts and 446 synthetic compounds that inhibited vaccinia virus DNAsynthesis, representing a hit rate of 1.6%. An extensive survey of thecurrent literature showed that the majority of these hit compounds werenever reported to function as inhibitors of DNA or RNA synthesis. Of thetotal 829 hit compounds, 178 were ranked as strong (greater than 70%inhibition), 271 were ranked as intermediate (50-70% inhibition), and380 were ranked as weak (30-50% inhibition) based on the colorimetricintensity of the read-out signal relative to the control signal obtainedwith no inhibitor (DMSO alone). Interestingly, the percentage of naturalextracts that inhibited DNA synthesis was greater than that of thesynthetic compounds. Likely, the DNA synthesis reaction is morevulnerable to the multitude of chemical entities present in naturalextracts than the singly purified compounds from synthetic libraries. Itis noted that the group of synthetic compounds with known biologicalactivities also had a greater hit rate than the synthetic compounds fromcombinatorial libraries. This is not surprising because many of theseknown compounds have been shown to modulate activities that utilize DNA(e.g., topoisomerization).

At this point, the inventor of the instant application decided to pursueonly the synthetic hit compounds because they have defined structuresand are available as purified chemicals. These synthetic hit compoundsrepresent a wide range of structures, which the inventor was able toclassify into 59 different chemical families. All of the strong and mostof the intermediate inhibitors from the combinatorial syntheticlibraries were confirmed to block vaccinia DNA synthesis upon manualretesting in the 96-well rapid plate assay. Follow-up studies wereconducted on 93 of the synthetic inhibitors from which all of thestructural families are represented. These compounds were evaluated forpotency in blocking vaccinia DNA synthesis in vitro, protecting culturedcells from infection with vaccinia virus, as well as cellular toxicity.

Potency of Hit Compounds in Blocking Vaccinia Virus DNA Synthesis InVitro

The inhibitory concentrations (IC50) for each of the 93 syntheticcompounds were determined. The assay was performed in 96-well platesusing conditions similar to those used for the primary HTS. Eachcompound, dissolved in DMSO, was tested in triplicate over a range ofconcentrations for inhibition of DNA synthesis catalyzed by vacciniaproteins. The percentage inhibition values were fitted on sigmoidaldose-response curves from which the IC50 was determined. Threerepresentative dose-response curves are presented in FIG. 14A. The IC₅₀values for 89 compounds ranged from 0.5 to 400 μM, while four compoundsshowed less than 50% inhibition at concentrations of 400 μM or higher(Tables 6 and 7).

Potency of Hit Compounds in Blocking Vaccinia Virus Infection In Vitro

The 93 DNA synthesis inhibitors were first tested in a plaque reductionassay to determine if they could prevent the formation of vaccinia virusplaques on BS-C-1 cell monolayers. For the plaque reduction assay, cellswere infected at a low multiplicity of infection (moi) to generateapproximately 50 vaccinia virus plaques in each well of a 48-well plate.Each compound was tested in triplicate over a range of concentrations,and the results were used to estimate the EC50, the effectiveconcentration of compound required to reduce the number of viral plaquesby 50%. The EC50 values for 75 compounds ranged from 5 to 200 μM, while18 compounds had no effect on the number of plaques (Tables 6 and 7).

TABLE 6 Summary of Activities of 16 Synthetic Hit Compounds DNA synthcell prot plaque red tox 20 h tox 72 h tox 120 h selectivity trappedcompd

IC

 (μM) EC

 (μM) EC

 (μM) TC

 (μM) TC₅₀ (μM) TC₅₀ (μM) index

by dsDNA 1 108 9.6 10 127 20 13.2 no 2 374 30 25 159 5.3 no 3 131 5050 >200 36 3 >4.0 no 4 12 50 15 157 40 3.1 no 5 129 45 15 115 5 2.6 no 630 95 75 >200 50 20 >2.1 no 7 26 100 75 >200 38 20 >2 no 8 52 10075 >100 >100 >100 >2 no 9 26 104 75 >200 40 20 >1.9 no 10 188 90 25 1661.8 no 11 43 130 75 >200 174 120 >1.5 no 12 32 80 75 >100 >100 50 >1.2no 13 46 100 50 >100 13 30 >1 no 14 75 188 150 >200 >200 50 >1 no 15 111236 100 >200 >200 75 >1 no 16 239 100 50 >100 >100 50 >1 no CDV 13250 >500 >500 300 >3.8 no

Refer to Chart 1 for the structures of compounds 1-16. ^(b)Theselectivity index was obtained as the ratio between TC₅₀ in the 20 hcytotoxicity assay and EC₅₀ in the 20 h cell protection assay.

indicates data missing or illegible when filed

TABLE 7 Structures and IC50 Values for 19 Synthetic Compounds that areStrong Inhibitors of Vaccinia DNA Synthesis in Vitro DNA SynthesisLibrary^(b) Compound Structure IC₅₀ (μM) 1

0.5 5

1.3 1

3.0 1

3.5 3

3.8 1

4.0 1

4.1 4

5.0 4

5.3 1

5.4 1

5.9 1

6.0 3

6.3 1

6.7 1

7.3 1

7.4 4

7.4 1

10 1

10 ^(a)These compounds do not have favorable antiviral activity incell-based assays because they are either highly toxic or failed toblock viral infection. ^(b)The synthetic compounds belong to thefollowing libraries: ChemDiv 3 (library 1); Maybridge 4 (library 3);ActiMol TimTec (library 4); and Biones 2 (library 5).

Next, the 93 compounds were tested in a cell protection assay. Thisassay was performed with amounts of virus capable of lysing every cellin the culture during the 20 h incubation period. Compounds withantiviral properties protect the cell monolayer from infection, asdetermined by the cellular mass, stained for absorbance at 570 nm. Eachcompound was tested in triplicate over a range of concentrations up to200 μM in 2-fold serial dilution increments. Dose-dependent plots weregenerated by fitting the results on four-parameter sigmoidal responsecurves. These plots were used to calculate for each compound theconcentration at which 50% antiviral protection is obtained relative tothe unprotected DMSO control (FIG. 14B). Twenty-nine of the 75 compoundsthat were active in the plaque assay also protected cells from viralinfection with EC50 values as low as 9.6 μM (Table 6).

While both the plaque reduction and the cell protection assays measurethe antiviral activity of compounds, they differ dramatically by theamount of virus used to infect the cell monolayer. Nevertheless, as seenin Table 6, there is an overall concordance between the EC50 valuesobtained with these two assays, confirming the efficacy of these hitcompounds in blocking viral infection.

Cellular Toxicity of Hit Compounds

The cell protection assay presented above indirectly measurescytotoxicity in that compounds that protect against viral infectionmust, a priori, perform this function without disrupting the cells.However, to directly assess the cytotoxicity of each of the 93compounds, the inventor of the instant application employed acytotoxicity assay that measures the metabolic activity of cells exposedto chemicals in the absence of virus. After culturing cells in thepresence of inhibitors, cell viability was determined byspectrophotometrically measuring the reduction of a water-solubletetrazolium salt to a dark-red formazan, which is catalyzed by amitochondrial activity. The percent cytotoxicity relative to the DMSOcontrol was fitted onto four-parameter sigmoidal dose-response curvesand the 50% toxic concentration values (TC50) were determined. After 20h, 15 compounds had negligible cytotoxicity even at the highest workingconcentration of 200 μM. Not surprisingly, longer incubation periods of72 and 120 h led to increased cytotoxicity (Table 7).

On the basis of the results of cell culture assays, the 93 compoundsfell into 3 groups: group I consisted of 18 compounds that had no effecton cells (Table 7); group II consisted of 46 compounds that inhibitedviral infection but were cytotoxic at similar concentrations (Table 7);group III consisted of 29 compounds that inhibited viral infection atconcentrations lower than the cytotoxic concentration (Chart 1, Table6).

Of note, the 18 synthetic compounds from group I did not have any effectin cell-based assays when tested at concentrations up to 200 μM: theydid not inhibit viral plaque formation, they did not protect cells fromviral infection, and they did not show any cytotoxicity. All of these 18compounds had tested positive in the rapid plate assay, some of thembeing very potent inhibitors of vaccinia DNA synthesis in vitro, withIC50 values as low as 1.3 μM (Table 7). This lack of antiviral activityand cytotoxicity is most probably due to the inability of thesecompounds to permeate the cell membrane. Chemical modifications thatincrease lypophilicity and cell permeability may confer antiviralactivity to these in vitro DNA synthesis inhibitors and convert theminto attractive lead compounds.

Selectivity Index of Hit Compounds

The selectivity index (SI) for each compound was calculated as the ratiobetween TC50 in the 20 h cytotoxicity assay and EC50 in the cellprotection assay. Six compounds had a SI of 4 or higher, with the mosteffective compound having a SI of 13, reflecting the ability to blockvaccinia virus infection at concentrations significantly lower thanthose producing cytotoxicity (Table 6). Of note, the SI for manycompounds is a conservative estimate because 50% cytotoxicity was notattained at the highest tested concentration of 200 μM. (e.g., compound3 in Table 6). As reference for antiviral activity, the inventor of theinstant application tested the well-known antipoxvirus drug CDV, whichfunctions as a chain terminator to inhibit viral DNA synthesis. CDV hada SI of greater than 4, which is in accord with the values previouslyreported. The compounds of the invention have potencies comparable toCDV, indicating their significance as potential poxvirus therapeutics.

Sixteen Hit Compounds Retain Their Antiviral Activity in the Presence ofa DNA Trap.

The compounds identified in our primary HTS were selected by theirability to block vaccinia virus DNA synthesis. Although many hitcompounds are likely inhibit DNA synthesis by interfering with aspecific mechanistic step (e.g., the catalytic incorporation of dNTP'sby DNA polymerase), the inventor was concerned that some hit compoundsbind to DNA (e.g., by intercalation), in which case they wouldnonspecifically inhibit DNA polymerases from incorporating nucleotides.To eliminate such nonspecific inhibitors, their antiviral activitieswere tested in the presence of large amounts of genomic double-strandedDNA added to the media of vaccinia-infected cells Because cells do nottake up exogenous DNA under normal culturing conditions, this DNA servesto trap nonspecific DNA binding compounds and disable their cellularuptake. Compounds that were affected by the exogenous DNA wereidentified by the loss of antiviral activity. As shown in Table 6, atotal of 16 compounds retained their antiviral activity in the presenceof the extracellular DNA trap, indicating that these compounds wererelevant inhibitors that block a mechanistic step in DNA synthesis.

When tested in 20 h assays, three relevant hit compounds had a SI of 4or higher, with the most effective compound having a SI of 13 (Table 6).These 16 compounds were tested for cell protection and cytotoxicity overlonger times (48 h and 72 h). At these longer times, the compoundsshowed diminishing antiviral activity and increasing cytotoxicity ascompared to the overnight assays. The antiviral activities of fourcompounds were high enough to allow calculation of EC50 values, but 12compounds showed less than 50% protection in the 72 h assays. Whencalculable, the SI for each compound was lower in the 72 h assay than inthe overnight assays. For example, compound I has an SI of 13.2 over 20h, and an SI of 3.2 over 72 h. The solubility of the 16 compounds wasverified in cell growth medium over the range of concentrations used inthe activity assays. Light scattering measurements indicated that 11compounds were completely soluble, while five compounds (8, 10, 12, 13,and 16) were just slightly insoluble at the EC₅₀.

The inventor of the instant application has discovered 16 inhibitors ofvaccinia DNA synthesis that have antiviral activity. These 16 inhibitorswere identified by the HTS of 49663 compounds using an in vitro DNAsynthesis rapid plate assay. All of the inhibitors effectively blockviral infection with minimal toxicity to the cells and could not betrapped outside the cells by exogenous DNA, indicating that theirantiviral activity is mediated by the disruption of an essential step inthe mechanism of viral DNA synthesis. Of particular interest, three ofthe inhibitors had selectivity indexes that approximate that of CDV, thewell-known poxvirus DNA synthesis inhibitor. Because these newinhibitors are not nucleoside analogues, they are expected to blockvaccinia DNA synthesis through a mechanism that is distinct from that ofCDV, a nucleoside analogue. Further development of these 16 syntheticcompounds could lead to useful pox antiviral compounds that willcomplement the inhibitory activity of CDV and thus reduce the emergenceof drug resistant mutants. On the basis of the high sequence similaritybetween the proteins of vaccinia and variola viruses that areresponsible for DNA synthesis, these new inhibitors can be equallyeffective against smallpox. Future enzymatic and virological studieswill identify the specific DNA synthesis proteins targeted by thesepoxvirus inhibitors.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to the precise embodiments, and that various changes andmodifications may be effected therein by those skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

1. A method of inhibiting, treating, or abrogating a poxvirus infectionin a subject comprising the step of administering to said subjectviridicatumtoxin (formula XLIV), NSC 55636, NSC 123526, or a combinationthereof whereby said viridicatumtoxin, NSC 55636, NSC 123526, or acombination thereof reduces, inhibits, or abrogates activity of a DNApolymerase of said poxvirus, thereby inhibiting, treating, or abrogatinga poxvirus infection in a subject.
 2. The method of claim 1, whereinsaid step of inhibiting replication of a poxvirus comprises inhibitingDNA synthesis.
 3. The method of claim 1, wherein said poxvirus is avaccinia virus or a variola virus.
 4. The method of claim 1, whereinsaid DNA polymerase is an E9 DNA polymerase or a homologue thereof froma different species.
 5. The method of claim 1, whereby said compoundreduces, inhibits, or abrogates interaction of said DNA polymerase witha processivity factor, thereby affecting activity of said DNApolymerase.
 6. The method of claim 5, wherein said processivity factoris an A20 or D4R processivity factor or a homologue thereof from adifferent species.
 7. A method of inhibiting, treating, or abrogating apoxvirus infection in a subject comprising the step of administering tosaid subject a compound having the formula I:A-X—B  I wherein A is:

or nothing Q is NCH₂CH₂R or O; R is OH, N(CH₃)₂ or CH₃; R₁ and R₂ areindependently, hydrogen, CH₃, OH, Cl; R₄, R₄′ and R₄″ are independently,hydrogen, COOH, OH, CF₃, Cl, Br, COOMe, OMe, N(CH₃)₂ or NO₂; W₁ isalkyl, alky-isothiourea or substituted alkyl B is

R₃ and R₃′ are hydrogen, COOH, OH, COOMe, Cl, CF₃, CH₃, OCH₃, N(CH₃)₂ orCN; W₂ is alkyl, alky-isothiourea or substituted alkyl, —SO2Et, H orisopropyl; W₃ is 2, 4 dimethoxy phenyl; W₄ is CH₃ or NH—W₃; and P ishydrogen, Fmoc, or Boc; X is nothing, NH, S,

—CO—, —CH₂S—, —N═CH— —COO—, —OCO—,

or A-X—B are fused rings, wherein X is a 5-membered substituted or notsubstituted heterocyclic or carbocyclic, optionally aromatic ringrepresented by one of the following structures:

A is

and B is

whereby said compound reduces, inhibits, or abrogates activity of a DNApolymerase of said poxvirus, thereby inhibiting, treating, or abrogatinga poxvirus infection in a subject.
 8. The method of claim 7, whereinsaid formula I is represented by the structure of

formula II: wherein: R1 is phenyl substituted or non substituted by OHor CH=(heterocyclic 5-10 membered ring); R₂ is O, NH, NR, wherein R isCH2CH2X; X is OH, CH₃, N(CH₃)₂; and R₃ and R₄ are independently H, Cl,CH₃ or OH.
 9. The method of claim 7, wherein said heterocyclic 5-10membered ring is


10. The method of claim 7, wherein said formula I is represented by thestructure of formula III:

wherein: A and B are independently hydrogen or they form a bond; X isNH, Net, C═NOH, CR₃R₄; R1′ and R1 are independently CF₃, COOH, COOCH₃,CH₃, Cl, OCH₃, OH or CH═N-triazole; R₂′ and R₂ are independently CF₃,COOH, COOCH₃, CH₃, Cl, OCH₃, OH or CH═N-triazole; and R₃ and R₄ areindependently chlorobenzene or SCH₂C(COOH)NH₂.
 11. The method of claim7, wherein said step of inhibiting replication of a poxvirus comprisesinhibiting DNA synthesis.
 12. The method of claim 7, wherein saidpoxvirus is a vaccinia virus or a variola virus.
 13. The method of claim7, wherein said DNA polymerase is an E9 DNA polymerase or a homologuethereof from a different species.
 14. The method of claim 7, wherebysaid compound reduces, inhibits, or abrogates interaction of said DNApolymerase with a processivity factor, thereby affecting activity ofsaid DNA polymerase.
 15. The method of claim 14, wherein saidprocessivity factor is an A20 or D4R processivity factor or a homologuethereof from a different species.
 16. The method of claim 7, whereinsaid compound is compound IV, V, VI, VII, VIE, IX, X, XI, XII, XIII,XIV, XV, XVI, XVII, XVIII, XIX, XX, XXII, XXIII, XXIV, XXV, XXVI, XXVII,XXVIII, XXIX, XXX, XXXI, XXXIII, XXXIV, XXXV, XXXVI, XXVII, XXXVIII,XXXIX, XL, XLII, XLII, or any mixtures thereof.
 17. A method ofinhibiting replication of a poxvirus, comprising the step of contactingsaid poxvirus with a compound having the formula XXI, XXXII, XLI, XLIVor any mixtures thereof, whereby said compound reduces, inhibits, orabrogates activity of a DNA polymerase of said poxvirus, therebyinhibiting replication of a poxvirus.
 18. The method of claim 17,wherein said inhibiting replication of a poxvirus comprises the step ofinhibiting DNA synthesis of said poxvirus.
 19. The method of claim 17,wherein said poxvirus is a vaccinia virus or a variola virus.
 20. Themethod of claim 17, wherein said DNA polymerase is an E9 DNA polymeraseor a homologue thereof from a different species.
 21. The method of claim17, whereby said compound reduces, inhibits, or abrogates interaction ofsaid DNA polymerase with a processivity factor, thereby affectingactivity of said DNA polymerase.
 22. The method of claim 21, whereinsaid processivity factor is an A20 or D4R processivity factor or ahomologue thereof from a different species.